Methods
Data reporting
No statistical methods were used to predetermine sample size. The
experiments were not randomized. The investigators were not blinded
to allocation during experiments and outcome assessment unless
otherwise stated.
Mice
Intestinal crypts, bile ducts and intestinal myofibroblasts were
extracted from 5–10-week-old heterozygous LGR5-eGFP-IRES-CreERT2
or wild-type C57BL/6J mice ( Jackson Laboratory), following animal
experimentation protocols prescribed by EPFL and the Federation for
Laboratory Animal Science Association (FELASA), in compliance with
local animal welfare laws, guidelines and policies.
Isolation of intestinal crypts
Mouse intestinal crypts and single LGR5–eGFP ISCs were isolated fol-
lowing previously described procedures. In brief, the proximal part of
the intestine was collected, opened longitudinally and washed with
ice-cold phosphate-buffered saline (PBS). The luminal side of the intes-
tine was scraped using a glass slide to remove luminal content and
villous structures. After a second wash with ice-cold PBS, the intestine
was cut into 2–4-mm pieces with scissors. The pieces were transferred
to a tube and further washed with cold PBS (5–10 times) with gentle vor-
texing. Intestinal fragments were incubated in PBS containing 20 mM
EDTA, for 20 min on ice. The supernatant was discarded and cold PBS
was added to the fragments. Crypts were released by manual shaking
of the suspension for 5 min. The supernatant was collected and passed
through a 70-μm strainer. The remaining tissue fragments were again
resuspended in cold PBS and triturated 5–10 times, and the supernatant
was passed through a 70-μm strainer. The previous step was repeated
once again. The three crypt-containing fractions were pooled together
and centrifuged at 110g for 5 min. The pellet was resuspended in cold
Advanced Dulbecco’s modified Eagle medium/Ham’s F-12 (Advanced
DMEM/F12) supplemented with 1× Glutamax, 10 mM HEPES and 100 μg
ml−1 penicillin–streptomycin (Gibco), and centrifuged at 84g to remove
single cells and tissue debris. Crypts were then cultured according to
the mouse intestinal organoids culture protocol (see ‘Cell culture’),
and after three passages single viable LGR5–eGFPhigh cells were sorted
by flow cytometry.
Cell culture
The following medium formulations were used in the protocol: base
medium (BM) was prepared from Advanced DMEM/F12 medium
supplemented with 1× Glutamax, 10 mM HEPES and 100 μg ml−1
penicillin–streptomycin (Gibco); BMGF medium was prepared
from BM supplemented with 1× B27 supplement, 1× N2 supplement
(Gibco) and 1 mM N-acetylcysteine (Sigma-Aldrich); ISC expan-
sion medium (ENRCV) was prepared from BMGF supplemented
with growth factors (50 ng ml−1 EGF (E) (Peprotech), 100 ng ml−1
Noggin (N) (EPFL Protein Expression Core Facility), 500 ng ml−1
R-Spondin 1 (R) (EPFL Protein Expression Core Facility)) and small
molecules (3 μM CHIR99021 (C) (Stemgent) and 1 mM valproic acid
(V) (Sigma-Aldrich)).
Isolated crypts or single cells were embedded in Matrigel (Corn-
ing; growth-factor-reduced, phenol-red-free formulation) and cast
into 25-μl droplets in a 24-well plate. After polymerization of Matrigel
(15 min, 37 °C), 500 μl of ENRCV medium was added. For freshly
extracted mouse crypts and single-cell culture, 2.5 μM Thiazovivin
(Stemgent) was included in ENRCV medium for the first two days to
prevent anoikis. Fresh medium was replenished every other day. For
passage, organoids were removed from Matrigel and mechanically
dissociated into smaller fragments, and then transferred to fresh
Matrigel. Passaging was performed every fourth day with a 1:4 split
ratio. Organoids were used for experiments between passage number
5 and 20. A detailed protocol describing organoid culture has been
deposited in the Protocol Exchange repository^28.
Intestinal myofibroblasts were isolated from C57BL/6J mice fol-
lowing a previously published protocol with slight modifications^29.
Mouse small intestinal tissue was processed and used for intestinal
crypt isolation as described above. Remaining intestinal fragments
were then washed and incubated in DMEM containing collagenase IV
(300 U ml−1; Invitrogen) and dispase (0.08 U ml−1; Roche) for 30 min
at 37 °C in a shaking water bath. The supernatant was then collected,
centrifuged at 280g for 5 min and resuspended in DMEM supplemented
with 10% heat-inactivated fetal bovine serum (HI-FBS), 100 μg ml−1
penicillin–streptomycin, 1× l-glutamine, 1× non-essential amino acids,
1× insulin–transferrin–selenium (Gibco) and 1× Primocin (InvivoGen).
Cells were then transferred to T75 culture flasks, and the medium was
changed after cell attachment (4–5 h) and every two days. Cells were
split 1:2 as needed and used between passage 3 and 8.
Collected liver tissues were minced and digested as previ-
ously described^30. In brief, whole liver was incubated in collagenase
XI (0.012%), dispase (0.012%) and FBS (1%) in Advanced DMEM/
F12 with 100 μg ml−1 penicillin–streptomycin, 1× Glutamax and
10 mM HEPES (Gibco), termed BM, for 2–3 h until bile duct frag-
ments were visible. Remaining tissue pieces were let to sediment
by gravitation and supernatant containing ductal fragments was
collected and centrifuged at 200 rpm for 4 min and washed with
PBS. Isolated fragments were resuspended in Matrigel (Corning;
growth-factor-reduced, phenol-red-free formulation) and cast
into 25-μl droplets in a 24-well plate. Following polymerization of
Matrigel, 500 μl of isolation medium, containing BM supplemented
with 1× B27 supplement, 1× N2 supplement (Gibco), 1.25 μM
N-acetylcysteine (Sigma-Aldrich), 10 nM gastrin (Sigma-Aldrich) and
growth factors (50 ng ml−1 EGF (Peprotech), 1 μg ml−1 R-Spondin 1 (EPFL
Protein Expression Core Facility), 100 ng ml−1 FGF-10 (Peprotech),
10 mM nicotinamide (Sigma-Aldrich), 50 ng ml−1 HGF (Peprotech),
1 μg ml−1 WNT3A (Time Bioscience), 100 ng ml−1 Noggin (EPFL Protein
Expression Core Facility) and 10 μM Y-27632 (Sigma-Aldrich) was added.
After four days of culture, the medium was changed to expansion
medium (EM) composed of isolation medium without WNT3A, Nog-
gin and Y-27632. Organoids were passaged by mechanical dissociation
every 10–14 days in split ratio 1:4 to 1:8.
The use of human embryonic stem (ES) cell lines was authorized
by the Office Fédéral de la Santé Publique (OFSP) after approval
by the cantonal ethical commission (CER-VD). All the experiments
reported in this Article were performed under authorization number
R-FP-S-2-0014-0000. HES3 MIXL1GFP/+ human ES cells were obtained
from A. G. Elefanty (Murdoch Children’s Research Institute) and rou-
tinely maintained in mTeSR1 medium (StemCell Technologies) on 6-well
plates coated with Matrigel human ES cell-qualified matrix (Corning).
The medium was changed every day. For routine culture the cells were
passaged every 4–5 days as small clumps using the Gentle Cell Dissocia-
tion Reagent (StemCell Technologies).
Macrophages were generated from HES3 MIXL1GFP/+ human ES cells
as previously described^31 .In brief, during the first two days of differ-
entiation, human ES cell colonies were specified to the mesoderm by
incubation in StemPro Medium (Gibco) with 5 ng ml−1 BMP4, 50 ng ml−1
VEGF (Peprotech) and 2 μM CHIR99021 (Stemgent). In the next step
(day 2–4) human haemangioblast-like cell formation was induced
by replacing CHIR99021 with 20 ng ml−1 hbFGF (Peprotech) and later
(day 4–6) maintained with VEGF and hbFGF only. The cells were then
cultured for the next 16 days in StemPro Medium for haematopoietic
stem cell differentiation with the following cytokines: differentiation
day 6: 10 ng ml−1 VEGF, 10 ng ml−1 hbFGF, 50 ng ml−1 SCF, 30 ng ml−1
DKK-1, 20 ng ml−1 IL-3, 20 ng ml−1 TPO and 20 ng ml−1 FLT3 (Peprotech);
differentiation day 8 and 10: 10 ng ml−1 VEGF, 10 ng ml−1 hbFGF, 50 ng
ml−1 SCF, 30 ng ml−1 DKK-1, 20 ng ml−1 IL-3, 20 ng ml−1 TPO and 20 ng