studies

544A AASLD ABSTRACTS HEPATOLOGY, October, 2015
676
Hepatic maturation of induced Pluripotent Stem Cells
is regulated by paracrine signals from endothelial and
mesenchymal stem cells in culture and during organoid
formation
Akihiro Asai 1 , Eitaro Aihara 2 , Tatsuki Mizuochi 1 , Kieran Phelan 1 ,
Christopher Mayhew 1 , Pranavkumar Shivakumar 1 , Takanori
Takebe 3 , James Wells 1 , Jorge A. Bezerra 1 ; 1 Pediatrics, Cincinnati
Children’s Hospital Medical Center, Cincinnati, OH; 2 Department
of Biology, University of Cincinnati, Cincinnati, OH; 3 Department
of Regenerative Medicine, Yokohama City University, Yokohama,
Japan
Background: To recapitulate key stages of organ development,
we engineered liver organoids using human induced pluripotent
stem cells (iPSC), human mesenchymal stem cells (MSC), and
human umbilical vein endothelial cells (HUVEC) as described
previously. Because regulatory mechanisms of organoid maturation
are not well defined, we investigated the differentiation
patterns of liver organoids and tested if differentiation of iPSC
requires direct contact with MSC and HUVEC. Methods: iPSC
were differentiated to hepatic endoderm (iPS-HE) by Act,Wnt3a,DMSO,and
Knock-Out Serum Replacement then co-cultured
with MSC and HUVEC to generate 3D organoids (liver
buds). Following implantation under kidney capsules of mice,
plasma levels of human albumin (ALB) and alpha1-antitrypsin
(A1AT) were monitored by ELISA. To investigate whether cellcell
contact is a key mechanism by which MSC and HUVEC
induce bud formation and maturation of iPS-hepatocytes,
we cultured iPS-HE in the upper well of a transwell system,
where MSC or HUVEC resided in the lower well individually
or in co-culture. The expression of alphafetoprotein (AFP),
HNF4a,ALB,A1AT,CPS1,BSEP,and CD31 was determined by
immunostaining or by ELISA. Results: Temporal-spatial analysis
by time-lapse microscopy revealed self assembly of iPS-HE,
MSC, and HUVEC into a 3D tissue in 24 hr following a condensation
and folding pattern to form a concave hemisphere.
Hepatocytic differentiation in the buds was demonstrated by
the AFP+/HNF4+ cells by whole-mount staining, in proximity to
CD31+ endothelial cells. After implantation of the buds, human
ALB (140-260ng/ml) and A1AT (62-174ng/ml) were detected
in mouse plasma. Explanted organoids showed hepatocyte-like
morphology organized in cellular clusters surrounded by vascular
networks. Using the transwell culture to determine the role
of paracrine effect, we found that ALB concentration in culture
supernatants achieved 1587±233ng/ml/day in iPS-HE with
MSC in the lower well (iPS/MSC) and 1881±162ng/ml/day
in iPS-HE/HUVEC, which were higher than iPS-HE cultured with
MSC+HUVEC in the lower (455±110ng/ml/day) or iPS-HE
alone (81±21ng/ml/day, P