studies

554A AASLD ABSTRACTS HEPATOLOGY, October, 2015
698
Decellularized Spleen Matrix for Regeneration of Functional
Hepatic-like Tissue by Reseeding Bone Marrow
Mesenchymal Stem Cells
Junxi Xiang 1,2 , Xinglong Zheng 1,2 , Lifei Yang 2 , Rui Gao 2 , Yi Lv 1,2 ;
1 Department of Hepatobiliary Surgery, First Affiliated Hospital
of Xi’an Jiaotong University, Xi’an, China; 2 Research Institute of
Advanced Surgical Technology and Engineering, Xi’an Jiaotong
University, Xi’an, China
Purpose This present study intends to regenerate hepatic-like
tissue by reseeding and inducing differentiation of bone marrow
mesenchymal stem cells (BMSCs) in decellularized spleen
matrix (DSM), in order to expand the donor organ pool for liver
transplantation. Methods The spleen, which provide access to
a wider range of alternative sources, was chosen for preparing
decellularized scaffold. We developed a physical-chemical
method, namely, repetitive freezing/thawing cycles, deionized
water, trypsin and Triton X-100 perfusion, to produce rats’
DSM scaffold. Then, 2×10 7 BMSCs were repopulated into
DSM for further dynamic culture and hepatic differentiation by
using a dened two-step inducing protocol. Two-dimensional
tissue culture asks were used as control. Results After decellularization,
the spleen matrix presented a biomimetic three-dimensional
porous architecture with intact vascular networks.
The native extracellular matrix proteins including collagen I,
collagen IV, bronectin and laminin were preserved, indicating
a similar three-dimensional microenvironment mimicking the
decellularized liver scaffold. When compared with two-dimensional
culture, the produced DSM bio-scaffold promoted the cell
engraftment and differentiation of BMSCs into hepatocyte-like
cells using a dened protocol during a 21-day differentiation
period, evidenced by morphological change, expression of
hepatic-associated genes and proteins, glycogen storage, indocyanine
green uptake, albumin secretion and urea production.
Conclusions The physical-chemical strategy was suitable for
producing decellularized spleen matrix, and the DSM scaffold
might have considerable potential in cell-based therapy and
fabricating functional hepatic-like tissue particularly because
the DSM can support hepatic differentiation of BMSCs and
effectively expand the donor pool.
Disclosures:
The following authors have nothing to disclose: Junxi Xiang, Xinglong Zheng, Lifei
Yang, Rui Gao, Yi Lv
699
Wnt/beta-catenin signaling activates dUTP pyrophosphatase,
a regulator of cellular dUTP levels, to prevent
uracil misincorporation into DNA in human liver cancer
stem cells
Yoshiro Asahina, Taro Yamashita, Hajime Takatori, Naoki Oishi,
Kouki Nio, Takehiro Hayashi, Yoshimoto Nomura, Tomoyuki
Hayashi, Mariko Yoshida, Tomomi Hashiba, Tsuyoshi Suda,
Masao Honda, Shuichi Kaneko; Disease Control and Homeostasis
Internal Medicine, Kanazawa University Hospital, Kanazawa,
Japan
BackgroundRecent evidence suggests that hepatocellular carcinoma
(HCC) is characterized by a hierarchical organization of
a subset of cells termed cancer stem cells (CSCs), which may
play a central role in resistance against cytotoxic reagents.
However, it is still unclear, mechanistically, how CSCs prevent
DNA damage induced by the reagents. In this study, we evaluated
the role of dUTP pyrophosphatase (dUTPase), known to
catalyze the hydrolysis of dUTP to dUMP, in the maintenance of
the dUMP pool and prevention of uracil misincorporation into
DNA in HCC CSCs. MethodsHuh7 and Huh1 cells were cultured
routinely in DMEM supplemented with 10% fetal bovine
serum. EpCAM-positive CSCs were purified by flow cytometry.
The expression levels of EpCAM and dUTPase were evaluated
by immunohistochemistry in 107 primary HCC samples that
were surgically resected. The promoter activity of DUT (encoding
dUTPase) was evaluated by a luciferase assay using Huh7
cells transfected with pGL3-DUT-full and pRL-SV40 plasmids.
The GSK3-beta inhibitor BIO, control MeBIO, and small interfering
RNAs (siRNAs) targeting scramble or CTNNB1 sequences
were used to regulate Wnt signaling in HCC cells. Results-
Sorted EpCAM-positive CSCs showed more nuclear accumulation
of dUTPase than EpCAM-negative Huh1 and Huh7 cells.
Immunohistochemical analysis indicated a strong positive correlation
between EpCAM and dUTPase expression in primary
HCCs. High-dUTPase HCCs showed lower overall survival than
that of low-dUTPase HCCs, and this difference was statistically
significant (P = 0.0012). BIO treatment increased, whereas
si-CTNNB1 treatment decreased the expression of EpCAM and
dUTPase compared with that of the control Huh7 cells. We
found a TCF4 binding element in the DUT promoter region,
and BIO treatment enhanced, whereas si-CTNNB1 treatment
attenuated DUT luciferase promoter activity compared with that
of the control cells. In contrast, although 5-fluorouracil treatment
enriched EpCAM-positive CSCs with nuclear accumulation of
dUTPase, it had no effect on DUT promoter activity, suggesting
no direct effect of 5-fluorouracil on DUT promoter activity. ConclusionTaken
together, our data suggest that Wnt/beta catenin
signaling activates dUTPase and may prevent DNA damage in
liver CSCs. dUTPase may be a good target to eradicate liver
CSCs resistant to cytotoxic reagents.
Disclosures:
Mariko Yoshida - Grant/Research Support: Bayer
Shuichi Kaneko - Grant/Research Support: MDS, Co., Inc, Chugai Pharma., Co.,
Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc,
Ajinomoto Co., Inc, Bristol Myers Squibb., Inc, Pfizer., Co., Inc, Astellas., Inc,
Takeda., Co., Inc, Otsuka„ÄÄPharmaceutical, Co., Inc, Eizai Co., Inc, Bayer
Japan, Eli lilly Japan
The following authors have nothing to disclose: Yoshiro Asahina, Taro Yamashita,
Hajime Takatori, Naoki Oishi, Kouki Nio, Takehiro Hayashi, Yoshimoto Nomura,
Tomoyuki Hayashi, Tomomi Hashiba, Tsuyoshi Suda, Masao Honda
700
Predictors of Response to Grazoprevir/Elbasvir Among
HCV Genotype 1 (GT1)–Infected Patients: Integrated
Analysis of Phase 2-3 Trials
Stefan Zeuzem 1 , Jürgen K. Rockstroh 2 , Paul Y. Kwo 3 , David Roth 4 ,
Eric Lawitz 5 , Mark S. Sulkowski 6 , Xavier Forns 7 , Janice Wahl 8 ,
Michael Robertson 8 , Bach-Yen T. Nguyen 8 , Eliav Barr 8 , Anita Y.
Howe 8 , Michael D. Miller 8 , Peggy Hwang 8 , Erluo Chen 8 , Kenneth
J. Koury 8 ; 1 Department of Internal Medicine I, J.W. Goethe University
Hospital, Frankfurt, Germany; 2 University of Bonn, Bonn,
Germany; 3 Indiana University School of Medicine, Indianapolis,
IN; 4 University of Miami Miller School of Medicine, Miami, FL;
5 Texas Liver Institute, University of Texas Health Science Center,
San Antonio, TX; 6 Johns Hopkins University School of Medicine,
Baltimore, MD; 7 Hospital Clinic, Barcelona, Spain; 8 Merck & Co.,
Inc., Kenilworth, NJ
Purpose: In phase 2-3 trials, 95% of GT1-infected patients
(±cirrhosis, HIV coinfection, prior treatment, end-stage renal
disease) who received grazoprevir 100 mg + elbasvir 50 mg
(GZR/EBR) ± ribavirin (RBV) achieved a sustained virologic
response 12 weeks after end of therapy (SVR12); 3% experienced
virologic failure. This analysis assessed potential predictors
of response to therapy. Methods: Analyses were performed
on 2 pooled datasets of 1408 GT1-infected patients enrolled