Cell News 04/2019
18
PRIZE WINNERS 2019
giocyte organoids depending on the culture medium used (Figure
3). This represents the first mouse embryonic culture system that
enables single hepatoblast to expand and commit to the two
liver epithelial derivatives (10).
Interestingly, we also have recently found that our liver or-
ganoid culture system (specially the adult) nicely recapitulates
many aspects of liver regeneration, in particular the molecular
mechanisms driving activation of the ductal compartment after
tissue injury
in vivo
(27). We have recently described that in
vitro, during the formation of liver organoids from differentiated
liver ductal cells, massive genome-wide changes on the expres-
sion profile occur to ductal cells when acquiring organoid state.
This recapitulates what occurs
in vivo
, upon damage, where
>1,500 genes become differentially expressed upon damage.
In fact, we found that >70% of all the differentially expressed
genes in vivo upon damage are also differential expressed
in
vitro
during the switch from differentiated to progenitor state
in organoids (1,108 out of 1,552 genes), thus revealing for the
first time that liver organoid cultures recapitulate most of the
transcriptional changes occurring during de-differentiation and
activation of adult ductal cells to a progenitor state
in vivo
(27).
Thus, our observations have validated the liver organoid model
as a faithful in vitro system where to study some aspects of liver
regeneration in a dish.
Liver organoids that recapitulate human adult
tissue and liver disease
While our mouse liver organoid culture system enabled the
long-term expansion of mouse liver cells and the study mouse
liver biology and regeneration in a dish, it could not support
the growth of human liver cells for more than 2–3 weeks.
Hence, in parallel studies to the above, we sought to establish
a human liver organoid culture system that robustly expanded
human liver cells in a dish. Gene expression profiles of human
liver cultures grown in ‘‘mouse liver medium’’ revealed that
Tgf-b signalling, a well-known inducer of growth arrest in the
liver, was strongly activated in the human cultures. Therefore,
to expand human liver cells in a dish we adapted our mouse
organoid culture system by inhibiting TGF
β
signalling and
activating cAMP signalling, a pathway known to induce prolif-
eration of biliary duct cells in vivo (28). Similarly to the mouse
counterparts, removal of the Wnt agonist Rspondin or blocking
Wnt secretion by porcupine inhibition resulted in rapid loss of
the cultures. Hence, Wnt signals, cAMP activation, and Tgf-b
inhibition resulted essential for long-term expansion of human
liver ductal cells (Figure 4A). Of note, both human and mouse
cultures could be established from single cells, which facilitated,
for the first time, the study of mutational processes in human
healthy tissue (9). These studies revealed that both, human and
mouse liver organoid cultures expand long-term while retaining
their differentiation capacity (Figure 4B) as well as their genetic
stability over time, since the base substitutions identified were
located in non-coding regions of the genome (29).
These advances in liver organoid technology have provided the
framework and array of models where to study human liver
development, maintenance and disease. In that regard, the
human liver suffers from many monogenic diseases, i.e., diseases
caused by mutations in single genes. While these are consid-
ered “rare” conditions, they account for 10 in every 1000 births
(30), represent a heterogeneous group of diseases. One of the
most frequent monogenic liver diseases is a1-antitrypsin (A1AT)
deficiency, an inherited disorder that predisposes to chronic ob-
structive pulmonary disease and chronic liver disease (31). A1AT
is secreted from the liver to protect the lung against proteolytic
damage from neutrophil elastase. The most frequent mutation
is the Z allele (Glu342Lys) of the SERPINA1 gene, which causes
accumulation of misfolded A1AT in hepatocytes. The ZZ mutant
phenotype is characterized by an 80% reduction of the protein
in plasma, which subsequently causes lung emphysema (31). We
grew organoids from 3 patients diagnosed with A1AT deficien-
cy and found that the organoids recapitulated the epithelial
counterpart of the disease, where A1AT protein aggregates were
found in the organoid cells, recapitulating the findings observed
in the histological sections of the patients (9). Similarly, organ-
oids obtained from an Alagille syndrome (AGS) patient, another
Figure 3: Mouse single cell bi-potent hepatoblasts generate
cholangiocyte and hepatocyte cells in culture
E9.5-E10 mouse embryo
Bi-potent Lgr5+
hepatoblast
Figure 4: Human liver organoids expand long term while preserving
ability to differentiate into hepatocytes and recapitulate disease in vitro
A) Long term self-renewal
B) Human liver functions
ZO1
Albumin
C) Liver cancer organoids derived
from cancer patient material
