Download Abstract Human fetal liver is the major site of haematopoiesis

Abstract
Human fetal liver is the major site of haematopoiesis throughout
gestation but at around 12 – 13, weeks hepatogenesis becomes
prominent. In the first trimester, the fetal liver contains a number of
putative stem cell populations in microenvironmental niches. This study
was designed to investigate these stem cell populations to determine the
origins of hepatogenesis in fetal life and to determine the implications for
hepatic regeneration in adults.
Mesenchymal stem cells were isolated from fetal liver samples (flMSC) on
the basis of adherence to tissue culture plastic. The phenotype of the
adherent population, using flow cytometry was determined as: CD29+ve,
CD105+ve, CD90+ve, CD73+ve, HLA ABC+ve, CD271-ve, CD45-ve,
CD34-ve, CD133-ve, CD11b-ve & HLA DR-ve. Differentiation of flMSC into
osteogenic, adipogenic and chondrogenic lineages was also demonstrated
as confirmation of the multipotential nature of these cells. Cells which
were expanded clonally showed the same characteristics as the primary
cell population. Thus, MSC isolated from human fetal liver conformed to
criteria stipulated by International Society of Cellular Therapy (ISCT).
flMSC were extensively passaged in vitro to relatively large passage
numbers (P15). It was observed that there was a reduction in expression
of CD105 associated with a decrease in proliferative and differentiation
properties of the cells. These findings appeared to be associated with
chromosomal abnormalities and a shortening of telomere length over
increasing passage number, which could be considered as a triggering of
cell senescence. It was concluded that the early passage numbers of
flMSC (P4-P8) are optimum for all studies and that CD105 is a reliable
and accessible surrogate marker for identification of senescent cells.
flMSC were cultured in proprietary medium, supplemented with growth
factors which are known to stimulate hepatogenesis. A greater degree of
hepatogenesis from flMSC was observed when the medium was
supplemented with oncostatin M (OSM), in comparison to culture in a
medium supplemented with fibroblast growth factor (FGF) or hepatocyte
growth factor (HGF). A hepatocyte-specific antibody and gene profiling
were used to analyse the development of flMSC in these culture
conditions.
Hepatic cluster formation (indocyanine green positive) was noted within 3
days when flMSC cultured in the presence of FGF. Then after subsequent
adding of OSM, cultured flMSC stained positively by flow cytometric
analysis using an optimised hepatocyte-specific antibody and gene
expression studies confirmed the presence of albumin, alpha-fetoprotein,
factor VIII and tryptophan 2,3 dioxygenase in these cells. The cells acquired a polygonal
morphology, similar to that of adult hepatocytes.
The flMSC characterised in this study were shown to possess
immunomodulatory activity: they do not induce a T-cell response and
they have an immunosuppressive effect on T-cells in mixed lymphocyte
culture. flMSC differentiated to hepatocytes have the same immunological
properties as undifferentiated flMSC, despite expression of HLA DR when
the cells are exposed to pro-inflammatory cytokines.
This study shows that human fetal liver-derived MSC can develop into
Functional hepatocytes in vitro and that these end-stage cells have
immunomodulatory properties. This de novo source of hepatic tissue
could have therapeutic utility with potential for transplantation across HLA
barriers.