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STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE
SOURCE
IDENTIFIER
Antibodies
mouse monoclonal anti-OCT3/4
BD Biosciences
Cat# 611202; RRID: AB_398736
rabbit polyclonal anti-SOX2
Abcam
Cat# ab97959; RRID: AB_2341193
rabbit monoclonal anti-SOX2
Abcam
Cat# ab93689; RRID: AB_10562630
mouse monoclonal anti-CDX2
Thermo Fisher Scientific
Cat# 39-7800; RRID: AB_2533435
mouse monoclonal anti-MUC5AC
Leica Biosystems
Cat# NCL-MUC-5AC; RRID: AB_442113
mouse monoclonal anti-MUC2
Leica Biosystems
Cat# NCL-MUC-2; RRID: AB_442112
rat monoclonal anti-E-cadherin
Takara
Cat# M108; RRID: AB_2895157
goat polyclonal anti-NANOG
R&D Systems
Cat# AF1997; RRID: AB_355097
goat polyclonal anti-SOX17
R&D Systems
Cat# AF1924; RRID: AB_355060
mouse monoclonal anti-a-SMA
Dako
Cat# M0851; RRID: AB_2223500
mouse monoclonal anti-b-III tubulin
Millipore
Cat# MAB1637; RRID: AB_2210524
mouse monoclonal anti-GATA4
Santa Cruz Biotechnology
Cat# sc-25310; RRID: AB_627667
mouse monoclonal anti-E-cadherin (E-cad)
Thermo Fisher Scientific
Cat# 33-4000; RRID: AB_2533118
goat polyclonal anti-E-cad
R&D systems
Cat# AF648; RRID: AB_355504
mouse monoclonal anti- cytokeratin 20 (CK20)
Dako
Cat# IR777; RRID: AB_2133718
mouse monoclonal anti- cytokeratin 7 (CK7)
Dako
Cat# M7018; RRID: AB_2134589
mouse monoclonal anti-CDX2
Biocare Medical
Cat# CM226-C; RRID: AB_2335616
rabbit monoclonal anti-SOX2
Thermo Fisher Scientific
Cat# MA5-16399; RRID: AB_2537918
goat polyclonal anti-PDX1
Abcam
Cat# ab47383 RRID: AB_2162359
rabbit polyclonal anti-Somatostatin
Dako
Cat# A0566; RRID: AB_2688022
mouse monoclonal anti-H, K-ATase
MBL International
Cat# D031-3; RRID: AB_590576
rabbit monoclonal anti-Synaptophysin
Roche
Cat# 760-4595; RRID: AB_2857955
mouse monoclonal anti-Chromogranin A
DAKO
Cat# M0869; RRID: AB_2081135
mouse monoclonal anti-b-actin
Sigma Aldrich
Cat# A5441; RRID: AB_476744
iMatrix-511
Nippi
Cat# 892-021
Stem Fit
Ajinomoto
Chemicals, peptides, and recombinant proteins
penicillin and streptomycin
Life Technologies
Cat# 15140-122
TrypLE Select
Life Technologies
Cat# A12177-01
Rock inhibitor(Y-27632)
WAKO
Cat# 253-00513
Fugene HD
Promega
Cat# E2311
G418
Nacalai Tesque
Cat# 16512-36
Activin A
Peprotech
Cat# 120-14-250UG
BMP4
Peprotech
Cat# 314-BP
B27
Life Technologies
Cat# 17504-001
L-glutamine
Life Technologies
Cat# 25030-081
FBS
BIOWEST
Cat# F7524
CHIR99021
TOCRIS
Cat# 4423
FGF4
Cell Guidance Systems
Cat# GFH31
NOGGIN
R&D Systems
Cat# 6057-NG
(Continued on next page)
14
iScience 25, 104314, May 20, 2022
iScience
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Article
OPEN ACCESS
Continued
REAGENT or RESOURCE
SOURCE
HEPES
Invitrogen
Cat# 15630-080
EGF
R&D Systems
Cat# 236-EG
Retinoic acid
WAKO
N2
Life Technologies
IDENTIFIER
Cat# 17502-001
Critical commercial assays
Prime Script II 1st strand cDNA Synthesis Kit
Takara
Cat# 6210B
Takara Ex Taq PCR kit
Takara
Cat# RR001A
TURBO DNA-free kit
Thermo Fisher Scientific
Cat# AM1907
TB Green Premix Ex Taq II
Takara
Cat# RR420A
LR clonase II
Life Technologies
Cat# 11791-020
XT ultraView Universal DAB Detection Kit
Ventana Medical Systems, Inc.
Cat# 760-500
Deposited data
RNA-seq
This paper
GEO: GSE173624
Listing genes highly expressed in the intestine
Fagerberg et al. (2014)
Array Express Archive
(www.ebi.ac.uk/arrayexpress/)
under the accession number E-MTAB-1733
the Human Protein Atlas
Karlsson et al. (2021)
https://www.proteinatlas.org
iPSC line (201B7)
Riken BRC (Tsukuba, Japan)
HPS0001, RRID:CVCL_A324
iPSC line (FF-PB-3AB4)
Suzuki et al. (2019)
iPSC line (CDX2-iPSC)
This paper
Experimental models: Cell lines
Colo320 DM
The Japanese Collection
JCRB0225, RRID:CVCL_0219
of Research Bioresources (JCRB)
Cell bank (Osaka, Japan)
Oligonucleotides
human CDX2 cloning primer, forward primer
This paper
50 -CACCATGTACGTGAGCTACCTCCTGGACAAGGAC-30
human CDX2 cloning primer, reverse primer 50 -TCAC
This paper
TGGGTGACGGTGGGGTTTAGCACCCCCCCAGTTG-30
Primers for hGAPDH, see Table S1
Okita et al. (2011)
Primers for hOCT3/4, see Table S1
Takahashi et al., 2007
Primers for hSOX2, see Table S1
Takahashi et al., 2007
Primers for hNANOG, see Table S1
Takahashi et al., 2007
Primers for hCDX2, see Table S1
This paper
Primers for hPDX1, see Table S1
This paper
Primers for hMUC5AC, see Table S1
This paper
Primers for hCK7, see Table S1
This paper
Primers for hCK20, see Table S1
This paper
Primers for hSOX17, see Table S1
This paper
Primers for hFOXA2, see Table S1
This paper
Recombinant DNA
pENTR/D-TOPO
Life Technologies
PB-TAC-ERN
Kim et al. (2016)
pCAG-PBase
Kim et al. (2016)
provided by Dr. Knut Woltjen
at Kyoto University
provided by Dr. Knut Woltjen
at Kyoto University
(Continued on next page)
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Continued
REAGENT or RESOURCE
SOURCE
IDENTIFIER
Software and algorithms
Strand NGS software program
JSTAT 6.5
Agilent
http://toukeijstat.web.fc2.com/
RESOURCE AVAILABILITY
Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by
the lead contact, Takashi Aoi (takaaoi@med.kobe-u.ac.jp).
Materials availability
This study did not generate new unique reagents.
Data and code availability
RNA-seq data have been deposited at GEO and are publicly available as of the date of publication.
This paper does not report original code.
Any additional information required to reanalyze the data reported in this paper is available from the
lead contact upon reasonable request.
EXPERIMENTAL MODEL AND SUBJECT DETAILS
iPSC culture
The validated iPSC line 201B7 was purchased from Riken Cell bank (Tsukuba, Japan) and transferred from
on-feeder to feeder-free conditions in our laboratory. We cultured the iPSC lines according to a previously
described method (Nakagawa et al., 2014). In brief, the culture plates were precoated with iMatrix-511
(0.5 mg/cm2), and the iPSC were maintained in Stem Fit medium (Ajinomoto) with penicillin (100 units/
mL) and streptomycin (100 mg/mL; Life Technologies, MA, USA) at 37 C with 5% CO2. The medium was
changed every other day and passaged every 7-10 days using 0.53 TrypLE Select (13 TrypLE Select diluted
1:1 with 0.5 mM EDTA/PBS [-]; Life Technologies) and Rho-associated kinase (Rock) inhibitor (Y-27632;
WAKO, Osaka, Japan).
METHOD DETAILS
Vector construction and generation of CDX2-iPSC
The cDNA encoding human CDX2 open reading frame (ORF) was amplified by polymerase chain reaction
(PCR) using the forward primer 50 -CACCATGTACGTGAGCTACCTCCTGGACAAGGAC-30 and reverse
primer 50 -TCACTGGGTGACGGTGGGGTTTAGCACCCCCCCAGTTG-30 , and the resulting PCR product
was cloned into pENTR/D-TOPO (Life Technologies) to generate pENTR-CDX2 clone according to the
manufacturer’s protocol. LR clonase II (Life Technologies) recombination was then performed using a
pENTR-CDX2 clone and the destination vector PB-TAC-ERN (Kim et al., 2016) to generate PB-TACCDX2-ERN.
To generate CDX2-iPSC, the dissociated single cells of FF-PB-3AB4 were seeded onto an iMatrix511coated 6-well plate at a density of 5 3 105 cells/well. The next day, the cells were transfected with
1.5 mg of pCAG-PBase (Kim et al., 2016) and 1.5 mg of PB-TAC-CDX2-ERN using Fugene HD (Promega,
WI, USA). Forty-eight hours after transfection, 100 mg/mL G418 (Nacalai Tesque, Kyoto, Japan) was added
to select transduced cells for 4 days. After 8 hiPSC colonies were isolated and expanded, the subclone
CDX2-iPSC that strongly expressed mCherry by DOX addition (1 mM for 3 days) was selected and used
in this paper. G418 (100 mg/mL) was continuously added during the maintenance culture of CDX2-iPSC
before differentiation into gastric organoids.
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Gastric organoid differentiation
Differentiation into gastric organoids was performed according to a previously reported method
(McCracken et al., 2014). In brief, 2.5 3 105 to 4.0 3 105 iPSC were plated as single cells in a 12-well dish
(Nunc Cell-Culture Treated Multidishes; Thermo Scientific, MA, USA) in StemFit medium with 10 mM of
Rock inhibitor Y-27632 (WAKO). The next day, the medium was changed to RPMI-1640 medium (Nacalai
Tesque) supplemented with 100 ng/mL of Activin A (Peprotech, NJ, USA), 50 ng/mL of BMP4 (Peprotech),
B27 (Life Technologies), L-glutamine (Life Technologies), penicillin and streptomycin. Activin A and B27
and L-glutamine were added for three days, and BMP4 was added on the first day. FBS (BIOWEST, Nuaille´,
France) contained increasing concentrations of 0%, 0.2 and 2.0% to induce differentiation into DE by Day 4.
To generate foregut spheroids, RPMI1640 medium supplemented with 2% FBS, 2 mM of CHIR99021
(TOCRIS, Bristol, UK), 500 ng/mL of FGF4 (Cell Gaidance, MO, USA), 200 ng/mL of NOGGIN (R&D Systems,
MN, USA), penicillin and streptomycin was added for 3 days until Day 7. Retinoic acid (2 mM; WAKO) was
added on Day 6.
For three-dimensional culture of gastric organoids, we used Matrigel (354234; CORNING, NY, USA),
advanced DMEM/F12 medium (Life Technologies), N2 (Life Technologies), B27, L-glutamine, 10 mM of
HEPES (Invitrogen, MA, USA), penicillin/streptomycin and 100 ng/mL of EGF (R&D Systems) at Day 7.
For the first 3 days, 2 mM retinoic acid and 200 ng/mL NOGGIN were added to the media. The media
were replaced every three to four days, as necessary.
A semi-quantitative or real-time quantitative reverse-transcriptase (RT)-PCR analysis
Total RNA was isolated using Trizol (Life Technologies) and treated with the TURBO DNA-free kit (Thermo
Fisher Scientific). For the extraction of RNA from CDX2-iPSC derived DOX(+)/() gastric organoids, Invitrogen Phasemaker Tubes (Thermo Fischer Scientific) were used with Trizol, and DNase treatment was not
performed. The Prime Script II 1st strand cDNA Synthesis Kit (Takara, Shiga, Japan) was used to synthesize
cDNA from 200-500 ng of total RNA.
For semi-quantitative RT-PCR analysis (Figures 1C, 2B, 3D, and S1B), the resulting cDNA was subjected to
PCR with a TaKaRa Ex Taq PCR kit (Takara). For the quantitative PCR analysis (Figures 4C and S2B), we
used a Light Cycler480 Real time PCR system (Roche) with TB Green Premix Ex Taq II (Takara). The
PCR primers used are listed in Table S1.
Western blotting
After 1 mM DOX treatment of CDX2-iPSC for 2 days, the cells were washed once with PBS, lysed with the
M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific) and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After the electrophoretic transfer of the proteins to the PVDF membranes, immunoblotting with mouse anti-CDX2 (Invitrogen) and mouse anti-b-actin (Sigma Aldrich, MO,
USA) followed by horseradish peroxidase (HRP)-conjugated secondary antibodies at a 1:3000 dilution in
Can Get Signal immunoreaction enhancer solution (TOYOBO, Osaka, Japan) was performed. The
Amersham Imager 600 imagers (Cytiva, Tokyo, Japan) was used to detect signals.
Immunocytochemistry
Cells were fixed with PBS containing 4% paraformaldehyde for 10 min at room temperature. After washing with
PBS, the cells were treated with 0.3% Triton X-100 in PBS for 45 min at room temperature and blocked with 2%
skim-milk in PBS for 1 h. The cells were incubated with primary antibodies at 4 C overnight and then stained with
secondary antibodies. The primary antibodies were mouse anti-OCT3/4 (611202, dilution 1:200; BD transduction
Laboratories, New Jersey, USA), rabbit anti-SOX2 (ab97959, dilution 1:100; abcam, Cambridge, UK), rabbit antiSOX2 (ab93689, dilution 1:100; abcam), mouse anti-CDX2 (39-7800, dilution 1:100; Invitrogen), mouse anti-MUC5AC (CLH2, dilution 1:100; Leica, Nussloch, Germany), anti-MUC2 mouse monoclonal antibody (Ccp58, dilution 1:200; Leica), rat anti-E-cadherin (M108, dilution 1:500; Takara), goat anti-NANOG (AF1997, dilution
1:200; R&D Systems), goat anti-SOX17 (AF1924, dilution 1:200, R&D Systems), mouse anti-a-SMA (M0851, dilution 1:200; Dako, Glostrup, Denmark), mouse anti-b-III tubulin (MAB1637, dilution 1:200; Millipore, MA, USA) and
anti-GATA4 mouse monoclonal antibody (sc-25310, dilution 1:200; Santa Cruz, TX, USA). All of the secondary
antibodies (Alexa Fluor 594-conjugated anti-mouse or anti-goat IgG, Alexa Fluor 488-conjugated anti-mouse,
anti-goat or anti-rat; or anti-rabbit IgG, Cy5 647-conjugated anti-mouse IgG) were obtained from Life
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Technologies. Hoechst 33342 (WAKO) was used for nuclear staining. In Figures 2D, 3B, 3E, 4E, 4F, 6B, S7B and
S11, representative data of two or three independent experiments are shown.
In vitro spontaneous differentiation via embryoid body formation
For embryoid body (EB) formation, undifferentiated iPSC were dissociated into single cells, resuspended in
Primate ES medium (Reprocell) containing 20 mM Rock inhibitor Y-27632 (WAKO) and seeded on low-celladhesion 96-well spindle-bottom plates (PrimeSurface, Sumitomo Bakelite, MS-9096M; Tokyo, Japan) at
1 3 104 cells per well. After 7 days of culture, the EBs were transferred to gelatin-coated 24-well plates
and cultured in the same medium for another 7 days. The differentiated cells were immune-stained with
the indicated antibodies.
Karyotype analyses
The G-band karyotype analysis for FF-PB-3AB4 was performed at Chromocenter, Inc. (Yonago, Japan).
Frozen section samples
The cultured organoids were fixed with 4% paraformaldehyde and then embedded in Tissue-Tek O.C.T
Compound (Sakura Finetek Japan, Tokyo, Japan) and frozen at 80 C. The frozen samples were sectioned
at 5-8 mm on a cryostat.
Histological and immunohistochemical analyses of t ...
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