Liver with end-stage chronic liver disease or acute

        Liver disease is one of the major reasons of
mortality worldwide, accounting for two million deaths every year. Liver, an
organ with greatest inbuilt regenerative capacity, cannot repair its damaged
tissues in end-stage liver diseases 1. The only therapeutic option currently for
patients with end-stage chronic liver disease or acute liver failure is still
liver transplantation, but it has serious limitations like donor availability
and organ rejection. Development of substantial regeneration
therapy for these liver diseases is an urgent task 2.

        Cellular therapy using stem cells looks
promising due to the unique plasticity and differentiation potential of stem
cells towards functional hepatocytes 3. Oval cells (resident hepatic stem
cells) are the best candidates to repopulate damaged liver, but they are miserly and are unfavorable for large-scale expansion
during clinical application 4. Embryonic stem cells possess massive
hepatogenic potential, but the risk of tumor formation and ethical issues
restrict their therapeutic application 5. Similarly, induced pluripotent stem
cells (iPSCs), though hold substantial promises
for hepatocyte generation, their effective and safe use in cell therapy is
still to be examined 6.

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          Adult stem cells from bone marrow and adipose
tissue are able to trans-differentiate into hepatocytes 7, 8, but the number of these cells
decreases significantly with age and the technique of their
collection remains invasive and is not free
from risks. This has directed to the evaluation of alternative sources from
embryonic derived tissue 9, 10.

          Stem cells from umbilical cord blood (UCB) are biologically young cells with a lot of
advantages, including absence of immunological maturity of adult stem cells, so they are
less rejected by human immune cells, and presence of high proliferation and
differentiation capacity with less risk of disease transmission. Moreover, the
collection process of cord blood is safe, easy and painless with possibility to
cryopreserve and revitalize the cells efficiently 11.

           Studies have revealed the presence of
different types of stem cells in UCB. Two major types of stem cells in UCB are
hematopoietic stem cells and mesenchymal stem cells (MSCs) 7. The
hepatocyte-like cells generated from UCB-derived MSCs submit an alternative
reservoir of functional hepatocytes which will give great benefits in liver
disease treatments, drug development including toxicological research, and
other medical applications 11. 

           Cellular therapy for end-stage liver
failures by human MSCs-derived
hepatocytes can be used safely and may improve the patient’s quality of life 12.
According to previously published protocols, hepatic trans-differentiation of human
MSCs is routinely accomplished by induction with multiple recombinant growth
factors and cytokines, which are very expensive 13. Several studies have
shown a high cost, time-consuming and complicated steps induction of MSCs into
hepatic lineage 14.

        Therefore
developing simple and effective protocol to generate functional hepatocyte-like
cells from MSCs is an urgent need and the study of hepatic
differentiation of stem cells is important from both the clinical application and basic science especially because there is an insisting need for
an adequate supply of human hepatocytes for transplantation 15.

        This study evaluates the potential of in vitro hepatic
trans-differentiation of human MSCs- derived from UCB by hepatocyte growth factor (HGF) and tries to discover
the best time for harvesting the differentiateded cells with good quality and
quantity.UCB is a rich source of MSCs 20. The capability to obtain effective MSCs-derived
hepatocytes would improve cell
therapy for liver diseases 24. We
successfully isolated and characterized MSCs from 11 samples of 30 (36.67%) with high viability and proliferation
capacity. In support of our findings, Denner et al. 25 succeeded to isolate UCB-derived MSCs from 16 samples of 42
(38.09%), and Kazemnejad et al. 26 separated MSCs from 4 of 11 cord blood samples (36.4%).  

         In this study, the cultured cells had specific characters of MSCs (they were spindle shaped cells attached to the
surface with high proliferation capacity). These
cells were positive for MSCs markers, including CD90 (41.3 ± 1.7 %) and CD105
(73.5 ± 3.8 %) but negative for hematopoietic markers, including CD34 and CD45. This was also
reported by Semedo et al. 27.

         Effective trans-differentiation
of MSCs towards hepatocyte-like cells in vitro was routinely achieved
using an array of recombinant growth factors HGF, fibroblast growth factor
(FGF), epidermal growth factor (EGF), cytokines Oncostatin M (OSM) and
chemical compounds (dexamethasone, nicotinamide, insulin etc.) either as a
cocktail or in a sequential manner 14. However, hepatic inductions
with multiple recombinant growth factors are not optimal for clinical
applications as it is expensive and time-consuming
technique. HGF is the key factor for liver growth and function. In our
study, we successfully differentiated UCB-derived MSCs into hepatocyte-like
cells by HGF and we tried to detect the best time for harvesting the maximum
number of functional hepatocytes.

         In the
present study, differentiation was evident by cytoplasmic contraction,
accumulations of cytoplasmic granules and change in fibroblastic morphology of
MSC to cuboidal, polygonal morphology of hepatocytes. The
morphological change and confluence of induced cells were time dependent and by the end of the day 20 differentiated
cells were very similar to hepatocytes appearance with the highest confluence.
The viability of induced MSCs was high along the culture duration, with a narrow
range (87%-98%). So, it didn’t significantly correlate with any of the measured
variables (culture duration, confluence, urea production and albumin
secretion).  

           Our data demonstrated that the number and
viability of induced cells increased significantly during culture till day 20 that
may be due to anti-apoptotic and anti-necrotic effect of HGF. The viability
of induced cells reached its maximum ratio after 20 days of induction and then
the viable cells decreased significantly with longer culture duration, which
may be due to decreased amount of nutrient and increased wastes formed by the
cells in the crowded media. It may also be that, the cells reached a maximum
number of divisions and were unable to divide anymore. This goes with the
results reported by Kang et al. 28, who found that hepatocyte-like
cells can be obtained from MSCs after 21 days
of culture in hepatogenic medium
with high number and viability. On the
other hand, Nonome et
al. 17 cultured human MSCs for 28 days
in hepatogenic medium containing HGF and FGF and found that the viability of
cultured cells decreased with time.

         In the current
work, The concentration of  urea
increased significantly on day 12  and reached
maximum concentration on day 20 then significantly decreased with longer
culture duration, which may be due to significant decrease in viability. Urea
production is significantly positively correlated with culture duration,
confluence of induced MSCs and albumin secretion. Our results were supported by
those demonstrated by Yang et al. 29 and
Wang et al. 30, who found that during differentiation of MSCs, urea production was detected in low level on day 12 then urea
production gradually increased until day
20 of induction.

            The immunocytochemical
analysis, herein, showed that the percentage of AFP-producing cells and
intensity of brown color increased significantly till reach maximum (45% ±
2.887) after 20 days of induction with HGF. These
results could be explained by the concomitant increase in the percentage
of  cells which showed
morphological transformation from bipolar
fibroblast-like morphology to round or oval-shaped cells, which reached ? 45%
on day 20. After day 20, the amount of
secreted AFP was decreased gradually with longer culture duration, which  may be attributed to the observed significant
decrease in viability of induced cells after day 20. Similar results were recorded by Tang et al. 31, who
studied the hepatic differentiation of human UCB stem cells by HGF and FGF and
found that the percentage of AFP producing cells increased with culture time
till day 21 at which about 50% of differentiated cells expressed AFP.

          In the present work, induced MSCs
expressed AFP and albumin after incubation with HGF in a time dependent manner.
The concentration of secreted albumin was significantly positively correlated
with culture duration, confluence of cells, and the amount of secreted urea. Quantitative gene expression study, herein, showed a significant
increase in gene expression of albumin (8.1 fold) on day 20, then a decrease on day 24 (6.3 fold), and a further decrease after 28
days of culture (5.8 fold). Our results can be interpreted by the work
of Bishi et al. 14, who reported that albumin
gene is a late hepatic gene which was produced in higher concentration in
mature hepatocytes than immature hepatocyte in developing stage.

Conclusion: Human
UCB-derived MSCs can differentiate into hepatocyte-like cells in vitro when
cultured in nutrient media containing HGF. These hepatocyte-like cells showed
high functional capacity as evidenced by albumin production, urea secretion and
AFP expression. The process of differentiation was time dependent with its peak
on day 20 of culture. Our study provides a simple and cheap strategy for in vitro
differentiation of human UCB-derived MSCs into hepatocyte-like cells during 20
day using HGF. 

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