1
DOI: 2016.097
Editorial Summary: This protocol describes the long term culture of liver and
pancreas 3D organoids from human and mouse, and differentiation of liver
organoids in vitro and in vivo. Methodology for genetic manipulation of these
self-renewing organoids is also detailed.
Culture and establishment of self-renewing human and mouse adult liver and
pancreas 3D organoids and their genetic manipulation
Laura Broutier
1*
, Amanda Andersson-Rolf
2*
, Christopher J Hindley
1*
, Sylvia F Boj
3
, Hans Clevers
4
, Bon-
Kyoung Koo
2,5$
, Meritxell Huch
1,2,6$
1. Wellcome Trust/Cancer Research UK Gurdon Institute, Henry Wellcome Building of Cancer and
Developmental Biology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.
2. Wellcome Trust - Medical Research Council Stem Cell Institute, University of Cambridge, Gleeson
Building, Tennis Court Road, Cambridge CB2 1QR, UK.
3. Foundation Hubrecht Organoid Technology (HUB), Upsasalaan 8, 3584CT Utrecht, The
Netherlands.
4. Hubrecht Institute-KNAW, University Medical Centre Utrecht, Uppsalalaan 8, 3584CT Utrecht, The
Netherlands.
5. Department of Genetics, University of Cambridge, Cambridge, CB2 3EH, UK.
6. Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge
CB2 3DY, UK.
*=equal contributions
$=corresponding author.
Correspondence:
m.huch@gurdon.cam.ac.uk
bkk25@cam.ac.uk
2
ABSTRACT
Adult somatic tissues have proven difficult to expand in vitro, largely due to the complexity of
recreating appropriate environmental signals in culture. We have overcome this problem recently
and developed culture conditions for adult stem cells that allow the long-term expansion of adult
primary tissues from small intestine, stomach, liver and pancreas into self-assembling 3D structures
that we have termed organoids. We describe a detailed protocol that describes how to grow adult
mouse and human liver and pancreas organoids; from cell isolation and long-term expansion to
genetic manipulation in vitro. Liver and pancreas cells grow in a gel-based extracellular matrix (ECM)
and a defined medium. The cells can self-organize into organoids that self-renew in vitro whilst
retaining their tissue-of-origin commitment, genetic stability and differentiation potential into
functional cells in vitro (hepatocytes) and in vivo (hepatocytes and endocrine cells). Genetic
modification of these organoids opens up avenues for the manipulation of adult stem cells in vitro,
which could facilitate the study of human biology and allow gene correction for regenerative
medicine purposes. The complete protocol takes 1-4 weeks to generate self-renewing 3D organoids
and perform genetic manipulation experiments and requires basic scientific training to perform.
KEYWORDS: organoid, liver, pancreas, genetic manipulation, stem cell culture
3
INTRODUCTION
Adult somatic tissue-resident stem cells are gaining much attention for their intrinsic ability to self-
renew and differentiate into cell types present in adult tissues, whilst retaining their genetic integrity
and commitment to their tissue-of-origin
1
. The recent development of 3D cell culture conditions for
adult organs has allowed the in vitro expansion of primary tissues from healthy adult mouse and
human tissue as well as from tissue affected by disease (patient-derived tissue). In parallel, the
development of human ESC- and iPSC-derived cultures that faithfully recapitulate the differentiation
of ectoderm (e.g., retina, brain)
2,3
and endoderm derivatives (stomach, small intestine, liver, lung or
thyroid [AU: Please include specific examples])
4,5
has opened up avenues to study human
development in vitro in an unprecedented manner.
In our previous studies we developed a 3D culture system that allows the long-term expansion of
small intestine
6
and stomach epithelial stem cells in organoid cultures
7
. Here, we describe recently
established protocols for the long-term expansion and genetic manipulation of adult liver and
pancreas cells in 3D organoid cultures from mouse and human models. We also explain the utility of
these organoid cultures in disease modelling
8-13
.
Development of the Protocol for Organoid Culture from Liver and Pancreas cells
The liver and pancreas develop from a common population of endodermal progenitors (reviewed in
ref 14). Although both organs share several aspects of their morphogenesis and development, they
give rise to very distinct functional organs with very different regenerative capacities
14
. The liver is
mainly formed of two types of epithelial cells, the hepatocytes and biliary epithelial cells (BECs; also
known as cholangiocytes), that, together with several non-epithelial cell types (stellate cells, stromal
cells, Kupffer cells and endothelial cells), generate the basic functional structure termed the hepatic
lobule
15
. The pancreas is formed of two functionally distinct compartments: the exocrine
compartment (comprising of duct and acinar cells) and the endocrine compartment (comprising of
the islets of Langerhans). The genes and molecular pathways regulating the embryonic development
of these two organs are evolutionarily conserved among all vertebrates studied. These include FGF,
HGF, Wnt, BMP, RA and TGFβ pathways that promote progenitor proliferation, migration and
survival
14,16-18
. Despite our knowledge of the signalling pathways involved during liver and pancreas
development, in vitro expansion of adult pancreas or liver cells has remained a challenge. It was
previously shown that primary liver cells could be maintained in culture in the presence of EGF, HGF
and the corticoid Dexamethasone
19
. However, these conditions did not allow the long-term
expansion of liver progenitors. Similarly, mouse embryonic pancreas cultures able to differentiate
into endocrine and exocrine lineages in vitro had been obtained from embryonic progenitor pools
20
or hESCs
21,22
. However, these systems do not allow the long-term expansion of pancreas cells in
vitro.
4
The knowledge acquired from developmental studies, combined with our own expertise in stem cell
cultures from adult small intestine
6
and stomach
7
, allowed us to define, for the first time, culture
conditions for the expansion and differentiation of mouse and human adult primary liver
8,9
and
pancreas
10,11
tissue. Thus, by providing an ECM (extracellular matrix) environment together with a
combination of growth factors essential during liver development and regeneration (HGF, EGF, FGF,
Rspondin-1)
8,15,23
, we developed a 3D in vitro culture system for the long-term expansion of adult
mouse and human liver cells. Likewise, using similar culture conditions as for the liver organoids, we
also established pancreas 3D cultures that allow the long-term expansion of adult mouse and human
pancreas ductal cells in vitro. For pancreas organoid cultures, Noggin, but not HGF, is a required
growth factor
10
.
Organoid cultures for mouse and human liver and pancreas tissue. To develop a long-term
expandable adult liver culture we combined Matrigel, which acts as the ECM, together with HGF,
EGF, FGF and Rspondin-1. Under these culture conditions both healthy adult mouse liver tissue and
damage-induced progenitors can be expanded for months in culture
8
(Fig. 1 & 2). The cellular
expansion is exponential, allowing one to obtain up to 10
6
cells from 1 single liver progenitor in ~5-6
weeks. The cells express a mixture of both ductal (Krt7, Krt19, Sox9, Epcam, MIC1-1C3) (Fig. 3A-B) as
well as hepatocyte (Ttr, HNF4α) markers
8
. Typically, the expanded cells will self-organize into 3D
structures that harbour a Krt19
+
, single-layered epithelial compartment resembling the hepatic
ductal compartment, and a pseudo-stratified compartment that partially resembles the liver bud of
an E9-E10 embryo
24
(Fig. 3B).
We have recently adapted this system to the culture of human material. We have reported that
single EpCAM
+
adult human liver cells expand long-term (>5months) in vitro as 3D organoid
cultures
9
. We achieved this by supplementing the medium with a small molecule which raises the
levels of cAMP, an activator of ductal proliferation
25
, and by blocking TGFβ signalling, which acts to
induce differentiation (Table 1). Thus, adult liver tissue from humans, but not mice, requires
regulation of TGFsignalling and cAMP activity for long-term expansion.
In contrast to the remarkable expansion capacity of liver organoid cultures, differentiation into
hepatocytes does not occur spontaneously. To differentiate the proliferative cells along the
hepatocyte lineage, we defined a differentiation medium. We found that blockade of ductal fate by
Notch inhibition, a potent inducer of ductal morphogenesis
26
, in combination with the removal of
Rspondin-1 and addition of Dexamethasone and BMP facilitated the differentiation of the organoids
to a hepatocyte fate, for both mouse and human (Fig. 3A-C)
8,9
. Shifting the cultures to this
differentiation medium not only upregulated the expression of classic hepatocyte markers
(cytochromes, albumin or Alpha-1-antitrypsin) but endowed the cells with functional hepatocyte
characteristics in vitro, such as albumin production, cytochrome activity or bile acid production. Of
note, when these hepatocyte-like cells were transplanted into a mouse model of Tyrosinaemia Type
I liver disease (FAH
-/-
mouse model)
8
or into an immunodeficient liver injury model with
CCl
4
/retrorsine
9
, the organoid cells engrafted into the liver tissue, generating clusters of functional
hepatocytes in vivo.
5
The pancreas organoids are entirely composed of ductal (Sox9
+
, Krt19
+
) cells that express the
embryonic progenitor marker Pdx1
27
. To expand pancreas ductal cells we used a similar protocol as
for liver organoids. Thus, adult pancreas organoid cultures will expand long-term in the presence of
EGF, Rspondin-1, FGF10 and Noggin
10
. So far, the direct differentiation of these cells along endocrine
lineages in vitro has proven unsuccessful. However, when adult pancreas organoid cells were mixed
with embryonic E13 mouse pancreas cells and immediately transplanted into the kidney capsule of
immunocompromised mice, the adult-derived organoid cells readily differentiated into fully mature,
mono-hormonal (insulin-, glucagon- or somatostatin-positive) cells in vivo
10
. Using similar culture
conditions, human pancreas duct cells can be expanded in vitro for 4-5 months
11
. However, their
endocrine differentiation potential has not been assessed yet.
Genetic manipulation in organoid cultures. Genetic manipulation has been of crucial importance in
understanding the biological function of numerous genes, and has provided knowledge of how
mutations in certain genes are the cause of diseases such as cancer and obesity
28-31
. We have
recently shown that gene transfer to organoids is feasible and allows the study of gene function as
well as gene targeting in vitro in 3D cultures
32
. For example, we successfully investigated the
function of RNF43, a novel E3 ubiquitin ligase, by retroviral overexpression in organoid cultures, and
so defined it as a negative regulator of the Wnt pathway
33
. Similarly, CRISPR gene targeting allowed
us to perform gene correction of the cystic fibrosis transmembrane conductor receptor (CFTR) in
intestinal organoids derived from cystic fibrosis patients
13
. These examples demonstrate how
retroviral gene overexpression and CRISPR/Cas-mediated gene editing can be used for studying gene
function, as well as for the potential application of autologous cell therapy.
Here we describe in detail two methods for gene transfer to liver and pancreas organoid cultures:
first, retroviral transduction
12
, which allows stable integration of the gene of interest into the
genome, and second, liposomal transfection allowing transient transfection
13
. Collectively, these
protocols will allow gene function analysis in murine and human organoid cultures by gene
overexpression, knockdown or transient transfection. First, organoids are trypsinized to single cells.
Following dissociation, retroviral transduction efficiency is further enhanced by the addition of
polybrene to the culture media which enhances virus adsorption to target cell membranes
34
. For
transfection, genes are delivered using a liposomal agent (Lipofectamine 2000). The single cells are
mixed with either virus or liposomal transduction or transfection mix and spin-inoculated. We have
found that spin-inoculation is not essential, however it increases the transduction or transfection
efficiency significantly. Following spin-inoculation, single cells are incubated in the transduction or
transfection mix. Organoids are amenable to transduction by retroviruses, lentiviruses and
adenoviruses
12,35,36
.
