studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 1145A
1921
3D culture strategies for improved MSCs-derived
hepatocyte-like cells: potential toxicological and clinical
applications
Madalena Z. Cipriano 1 , Nora Freyer 2 , Fanny Knoespel 2 , Rita Barcia
3 , Pedro E. Cruz 3 , Helder Cruz 3 , Nuno G. Oliveira 1 , Jorge M.
Santos 3 , Katrin Zeilinger 2 , Joana P. Miranda 1 ; 1 CBT, Research
Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade
de Lisboa. Av. Prof. Gama Pinto, 1649-003 Lisbon,
Portugal, Lisboa, Portugal; 2 Bioreactor Group, Berlin Brandenburg
Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin
Berlin, 13353 Berlin, Germany, Berlin, Germany; 3 ECBio
S.A., Rua Henrique Paiva Couceiro, N 27, 2700-451-Amadora,
Portugal, Amadora, Portugal
Background: Three-dimensional (3D) cultures have emerged
as promising alternative models for maintaining hepatocyte
phenotype in short and long-term in vitro cultures by better
resembling the in vivo environment of the liver. However,
human primary hepatocytes have limited availability and
high inter-individual differences. As such, the differentiation
of human stem cells into hepatocyte-like cells (HLCs) has been
suggested. Human neonatal mesenchymal stem cells (hnMSCs),
derived from the umbilical cord tissue, are particularly interesting
given their proliferation potential, low immunogenic
and immuno-modulatory properties along with a demonstrated
HLCs differentiation in monolayer cultures. However a mature
hepatic phenotype was not yet achieved. In this study, 3D cell
cultures, namely a spheroid (SC) and a 3D multi-compartment
membrane bioreactor (BR) system, were tested as an approach
for improving HLCs maturation in vitro. Methods: A 27-day
differentiation protocol consisting of the sequential addition
of cytokines/growth factors was optimized. Firstly, hnMSCs
(UCX ® ) were differentiated into hepatoblast-like cells in 2D for
17 days and afterwards cultured under 3D conditions (SC and
BR) or maintained in 2D as control for maturation over 10
days. Results: All systems enabled hnMSC differentiation into
HLCs as shown by positive immunostaining of hepatic markers,
such as cytoskeleton proteins (CK-18), the transcription
factor HNF-4α, albumin, the hepatic transporters OATP-C and
MRP-2 and CYP1A2 and CYP3A4. Additionally, the three culture
models displayed relevant glucose metabolism, tested by
the capacity to store glycogen and to release glucose into the
medium, and the ability to convert ammonia into urea, to produce
albumin and to perform phase I metabolism. However,
urea production (5.8 and 8.3 fold induction for the BR and the
SC, respectively) and EROD activity (4.8 and 3.8 fold induction
in the BR and the SC, respectively) were both increased in
3D relative to 2D. Albumin production was also induced 2.9
fold in the BR when compared to 2D. Finally, cells differentiated
within BR presented relevant CYP1A2 and 3A4 activities
when compared with 2D, whereas HLCs from spheroids and
2D presented similar CYP3A4, 1A1 and 2C9 activity on day
27. Conclusions: Overall both the SC and BR models improved
HLCs maturation suggesting its potential for in vitro and clinical
applications, namely drug toxicity prediction, regenerative
medicine or external liver support. Additionally, differential
HLCs phenotypes were observed for each of the 3D models,
further supporting that the optimal culture system should be
selected depending on the scientific purpose.
Disclosures:
Rita Barcia - Management Position: ECBio
Pedro E. Cruz - Board Membership: ECBio, S.A.
Helder Cruz - Board Membership: ECBio
Jorge M. Santos - Employment: ECBio
The following authors have nothing to disclose: Madalena Z. Cipriano, Nora
Freyer, Fanny Knoespel, Nuno G. Oliveira, Katrin Zeilinger, Joana P. Miranda
1922
Death and Liver Transplantations Occurring Within Two
Years of Drug-Induced Liver Injury
Paul H. Hayashi 1 , Don C. Rockey 2 , Robert J. Fontana 3 , Hans L.
Tillmann 4 , Neil Kaplowitz 5 , Huiman X. Barnhart 6 , Jiezhun Gu 6 ,
Averell H. Sherker 7 , Naga P. Chalasani 9 , Victor J. Navarro 8 ,
Jawad Ahmad 10 , Jay H. Hoofnagle 7 ; 1 Division of Gastroenterology
& Hepatology, University of North Carolina, Chapel Hill, NC;
2 Medical University of South Carolina, Charleston, SC; 3 University
of Michigan, Ann Arbor, MI; 4 East Carolina University, Greenville,
NC; 5 University of Southern California, Los Angeles, CA; 6 Duke
University, Durham, NC; 7 National Institutes of Health, Bethesda,
MD; 8 Einstein Medical Center, Philadelphia, PA; 9 Indiana University,
Indianapolis, IN; 10 Mount Sinai Medical Center, New York,
NY
Background: While idiosyncratic drug-induced liver injury (DILI)
is usually reversible, it can lead to liver transplantation (LT)
and/or death. But the overall mortality rate and risk factors for
death or LT are not well defined. Aim: To characterize the role
and clinical features of DILI in deaths and LT cases. Methods:
All patients in the Drug Induced Liver Injury Network (DILIN)
who died or underwent LT within 2 years of follow-up were
identified. Each case was reviewed independently by 2 hepatologists
who judged whether DILI had a primary, contributory
or no role in the death or LT. Cases of DILI having a primary
role were categorized as acute liver failure (ALF: 6 months) or other.
Cases of DILI having a contributory role were assigned another
cause of death (e.g. cancer, sepsis). Discrepancies between
reviewers were resolved by conference call. R-values (ALT/ULN
÷ ALP/ULN) at DILI onset were used to classify injury patterns
as hepatocellular (R>5), mixed (R 2-5) or cholestatic (R