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12.3 Liver Buds
Despite current efforts to produce mature cells, generated cells have poor function,
and even with functional hepatocytes, the therapeutic efficacy of cell-based inter-
ventions remains controversial, as shown in several clinical trials using human adult
hepatocytes. Therefore, in vitro generation of complete functional organs offers an
alternative to organ transplantation.
In our studies of vascularized organs such as the liver, we assumed that func-
tional cell differentiation from iPS cells may not occur in the absence of stromal
cellular interactions. Adult liver tissues comprise about 60% parenchymal hepatic
cells and 40% non-parenchymal stromal cells such as liver sinusoidal endothelial
cells, mesenchymal stellate cells, and Küpffer cells. Accordingly, spatiotemporal
control of dynamic intercellular interactions is essential for efficient and proper cell
differentiation from stem cells (Kamiya et al. 1999 ; Kinoshita et al. 1999 ; Tanimizu
et al. 2007 ; Suzuki et al. 2008 ; Okabe et al. 2009 ). However, we have so far been
unable to construct large three-dimensional multicellular tissues from pluripotent
stem cells on culture dishes. Because liver development starts with the formation of
hepatic endoderm from a thin layer of foregut endoderm, subsequent cellular inter-
actions between endoderm cells, endothelial cells, and mesenchymal cells are
essential for the formation of liver buds. Moreover, these interactions are estab-
lished prior to blood perfusion, which is likely essential to further development of
liver buds.
Previously, we attempted to mimic early organogenesis and recapitulate organo-
genic cellular interactions by generating three-dimensional liver buds from pluripo-
tent stem cells in vitro (Fig. 12.4) (Takebe et al. 2012 , 2013 , 2014 ). These liver buds
could be transplanted, and subsequent blood perfusion may facilitate maturation to
functional liver tissue. Thus, to mimic cellular interactions, we cocultured iPSC-
derived hepatic-specified endoderm, mesenchymal stem cells (MSCs), and human
umbilical vein endothelial cells (HUVECs) under various conditions. These studies
showed that, under defined conditions, large-scale morphogenetic changes were
induced by the interactions between these cell types. In particular, condensation of
the heterotypic cell mixture led to the formation of a large cellular mass and was
initiated by MSCs that depend on substrate stiffness. Hence, mesenchymal cell-
driven cellular contraction is essential for the dynamic condensation of heteroge-
neous cell mixtures.
In subsequent experiments, it appeared that these self-organized cellular masses
resembled the proliferation and cellular context of the liver buds observed in mouse
embryos. Moreover, transcriptome analyses of these iPSC-LBs revealed similar
expression profiles to those in embryonic day (E) 10.5 murine liver buds. The tim-
ing and stages during differentiation of iPSC into hepatocytes may be critical to
optimizing the functional advantages of liver buds. Potentially, further analyses of
iPSC-LB are required to reveal more details of the human hepatic differentiation
processes and may indicate dynamic conformational changes of monolayer gut
endoderm into three-dimensional LB. In addition, comparative analyses of tran-
K. Sekine et al.