studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 549A
687
Expression pattern of the stem/progenitor cell marker
Neighbor of Punc E 11 in different mouse models of
liver regeneration.
Vera Hoffmann, Andrea Bowe, Harald M. Curth, Tobias Goeser,
Dirk Nierhoff; University Hospital of Cologne, Department of Gastroenterology
and Hepatology, Cologne, Germany
Background: Regeneration after acute liver injury naturally
arises from the compartment of parenchymal liver cells. However,
if proliferation capacity of hepatocytes is inadequate,
liver progenitor cells are activated and can give rise to mature
hepatocytes and cholangiocytes. In this project, we have
focused on the expression pattern of the novel stem/progenitor
cell marker Neighbor of Punc E 11 (Nope) in liver regeneration
after acute and chronic liver injury. Methods: C57Bl6 mice on
normal chow, on a 3,5-diethoxycarbonyl-1,4-dihydro-collidin
(DDC) or a choline-deficient, ethionine-supplemented (CDE)
diet were investigated. DDC and CDE diet were optionally
followed by a regenerative period on normal chow. For additional
acute liver injury, subgroups of normal mice as well as
mice on a DDC diet were subjected to a partial hepatectomy
(PH) with or without retrorsine to block hepatocyte proliferation.
After defined time periods between 3 to 6 weeks, mice were
sacrificed and quantitative RT-PCR and immunohistochemical
costainings of the liver tissue for Nope with CK19, A6, EpCAM
(oval cell markers), E-cadherin or HNF4α (hepatocyte markers)
were performed. Results: Partial hepatectomy with or without
retrorsine did not induce any expression of Nope above background
level in the adult liver. After DDC diet the expression
level of Nope was increased in the RT-PCR. In the CDE model,
the expression level of Nope even reached the same level as
in the fetal liver. We were able to detect ductular proliferations
with coexpression of CK19, A6 and EpCAM with Nope.
In the DDC model, only A6 also stained positive in a minor
cell fraction within periductular Nope positive hepatocytic cell
populations. After PH in the DDC model, followed by normal
chow, the Nope expression level decreased, but small hepatocytic
cells in proximity to ductular structures stained positiv for
Nope. In the CDE model, we were able to detect clearly confined
Nope positive hepatocytic cell clusters mainly in proximity
to Nope positive ductular structures. Clearly distinguishable
from these Nope expressing cell populations, expression of
Nope was infrequently induced in HNF4α positive parenchymal
hepatocytes. Discussion: The oncofetal marker Nope is
expressed in CK19, A6 and EpCAM positive ductular cells as
well as in CK19 negative periductular small hepatocytic cells
presumably representing early hepatocytes after liver injury.
These Nope positive early hepatocytes can form confined
regenerative clusters with a minor fraction staining positive for
A6. In conclusion, Nope is a marker for progenitor cells and
periductular early hepatocytes in different mouse models of
liver regeneration.
Disclosures:
Tobias Goeser - Advisory Committees or Review Panels: Gilead, Johnson, BMS,
BMS; Grant/Research Support: Roche; Speaking and Teaching: Essex, BMS,
Gilead, Novartis, Falk, Roche, Essex, BMS, Gilead, Novartis, Falk
Dirk Nierhoff - Consulting: AbbVie; Speaking and Teaching: Gilead, AbbVie,
BMS, Janssen, MSD
The following authors have nothing to disclose: Vera Hoffmann, Andrea Bowe,
Harald M. Curth
688
Ex vivo-expansion of human CD34 + cells from patients
with liver cirrhosis enhances therapeutic efficacy of cell
transplantation for rat cirrhotic liver
Toru Nakamura 1,2 , Hironori Koga 1,2 , Hideki Iwamoto 1,2 , Yu
Ikezono 1,2 , Fumitaka Wada 1,2 , Mitsuhiko Abe 1,2 , Takahiko
Sakaue 1,2 , Takato Ueno 3,2 , Takuji Torimura 1 ; 1 Division of Gastroenterology,
Department of Medicine, Kurume University School of
Medicine, Kurume, Japan; 2 Liver Cancer Division, Research Center
for Innovative Cancer Therapy, Kurume University, Kurume, Japan;
3 Asakura Medical Association Hospital, Asakura, Japan
Background: We demonstrated that the transplantation of
human CD34 + cells into an immunodeficient rat liver fibrosis
model reduced liver fibrosis by suppressing activated hepatic
stellate cells and increasing MMPs activity, and led to hepatic
regeneration. Recently, we reported that autologous granulocyte-colony
stimulating factor (G-CSF)-mobilized CD34 + cell
transplantation for patients with decompensated liver cirrhosis
(LC) had therapeutic potential, but the colony-forming ability
of CD34 + cells from patients with decompensated LC was
reduced. Thus, recovery of CD34 + cell function is indispensable
for cell transplantation therapy of patients with LC. The aim of
this study was to investigate the efficacy of cell transplantation
therapy with ex vivo-expanded human CD34 + cells for carbon
tetrachloride (CCl 4
)-induced liver fibrosis model. Methods:
Human G-CSF-mobilized CD34 + cells of patients with LC were
isolated by magnetic cell sorting system. Recipient nude rats
were injected intraperitoneally with CCl 4
twice weekly for 3
weeks before initial treatment. Then, saline, 5×10 4 , or 1×10 6
non-expanded and expanded CD34 + cells/kg body weight
were transplanted via spleen, respectively. The administration
of CCl 4
was continued for three more weeks until the rats were
sacrificed. Examination items were as follows: 1) FACS and
Real-Time PCR analysis of non-expanded and expanded CD34 +
cells, 2) morphometry of fibrotic areas by Azan-Mallory staining,
3) immunohistochemistry using anti-CD31, smooth muscle
myosin heavy chain-1 (SM1), αSMA, Ki67, and PCNA antibodies,
and 4) the RT 2 Profiler TM PCR Array analysis. Results:
For 7 days, CD34 + cells were effectively expanded. Increased
expression of VE-cadherin, KDR and Tie-2 was determined by
FACS analysis. The expression of pro-angiogenic growth factors
in expanded CD34 + cells increased compared with non-expanded
CD34 + cells. The transplanted cells differentiated into
CD31 + and SM1 + cells. Expanded CD34 + cell transplantation
had dose-dependently reduced liver fibrosis. Assessments
of hepatocytes and sinusoidal endothelial cells proliferative
activity indicated the superior potency of expanded CD34 +
cells over non-expanded CD34 + cells. The PCR Array analysis
against the adhesion molecules was showed that the expression
of integrin αvβ3 was the most up-regulated gene compared
with before culture. Three weeks of treatment with Cilengitide,
which specially inhibits integrin αvβ3 signaling, resulted in the
inhibition of CD34 + cells migration and worsened liver fibrosis
in a dose-dependent manner. Conclusion: These findings suggest
that expanded CD34 + cell transplantation promote better
therapeutic effects for liver cirrhosis.
Disclosures:
The following authors have nothing to disclose: Toru Nakamura, Hironori Koga,
Hideki Iwamoto, Yu Ikezono, Fumitaka Wada, Mitsuhiko Abe, Takahiko Sakaue,
Takato Ueno, Takuji Torimura