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Generation of a vascularized and functional human liver from an iPSC-derived organ bud transplant

Authors:
  • Rutgers University School of Public Health

Abstract and Figures

Generation of functional and vascularized organs from human induced pluripotent stem cells (iPSCPSCPSCs) will facilitate our understanding of human developmental biology and disease modeling, hopefully offering a drug-screening platform and providing novel therapies against end-stage organ failure. Here we describe a protocol for the in vitro generation of a 3D liver bud from human iPSCPSCPSC cultures and the monitoring of further hepatic maturation after transplantation at various ectopic sites. iPSCPSCPSC-derived specified hepatic cells are dissociated and suspended with endothelial cells and mesenchymal stem cells. These mixed cells are then plated onto a presolidified matrix, and they form a 3D spherical tissue mass termed a liver bud (iPSCPSCPSC-LB) in 1–2 d. To facilitate additional maturation, 4-d-old iPSCPSCPSC-LBs are transplanted in the immunodeficient mouse. Live imaging has identified functional blood perfusion into the preformed human vascular networks. Functional analyses show the appearance of multiple hepatic functions in a chronological manner in vivo.
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© 2014 Nature America, Inc. All rights reserved.
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396 | VOL.9 NO.2 | 2014 | NATURE PROTOCOLS
INTRODUCTION
Potential of generative medicine, using iPSCs for treating organ
failure
Organ transplantation is the only curative method for treating
end-stage organ failure. Over the past decade, there has been
increased demand for organ transplantation throughout the
world owing to the increased incidence of organ failure. There
remains a shortage of organs relative to the existing demand; for
example, in the United States, 20,000 patients are actively wait-
ing for a donor liver for transplantation to treat end-stage liver
disease1. However, owing to the shortage of donor organs, only
7,000 liver transplants per year are performed in the United
States2. In the hope of eliminating this organ shortage, many
researchers are attempting to provide alternative approaches to
traditional solid-organ transplantation. By using iPSCs, it is now
possible to obtain patients’ own or immunocompatible pluripo-
tent stem cells that are capable of differentiating into functional
cells that could potentially compensate for the function of dam-
aged organs3. iPSCs can be directed to differentiate into various
specialized cell types with potential applications in the replace-
ment of diseased or damaged tissues.
Methods to differentiate iPSCs into functional cells
Over the past decade, stem cell research has made substantial
progress in improving our understanding of the various molecu-
lar mechanisms that control cellular differentiation, including
cell-type specification and reprogramming4. Numerous publica-
tions have described protocols that can induce the differentiation
of cells, with gene expression and functional profiles that resem-
ble those of terminally differentiated cells. By using combina-
tions of inductive or reprogramming factors, the direct in vitro
differentiation of PSCs into cells such as hepatocytes, insulin-
producing cells or dopaminergic neurons has been described5–9.
However, although the results have generally been impressive, no
protocol has successfully generated fully functional cells, such as
hepatocytes4 or beta cells, that possess a function comparable
to primary human parenchymal cells6. More importantly, con-
ventional cell- or tissue-based therapeutic approaches without
functional vasculature may lead to insufficient or inappropri-
ate engraftment after transplantation. Establishing methods to
grow vascularized organs from stem cells should lead to more
physiological engraftment. However, recapitulation of the spa-
tiotemporal control of the multicellular interactions that occur
during embryogenesis in stem cell cultures to generate a complex
and vascularized organ such as the liver has been impractical10.
The generation of organs via an iPSC-derived organ bud
transplant
As a starting point to generate a complex and vascularized
organ, we focused on the most primitive process of organogen-
esis because of its simplicity. During the earliest stage of liver
organogenesis, hepatic-specified endodermal cells delaminate
from the foregut endodermal sheet and form a 3D tissue termed
the liver bud, a condensed tissue mass that is quickly vascular-
ized11. Pioneering studies revealed that the formation of a 3D liver
bud depends on the exquisite orchestration of signals between
endodermal epithelial, mesenchymal and endothelial progeni-
tors before blood perfusion12. When trilineage cells were mixed
and plated on a noncoated plate such that the cells could not
adhere, there was 3D self-organization of the cells13. After further
refinements to our specific culture conditions, these organoge-
netic cellular interactions initiated 3D liver bud morphogenesis
from hepatic endodermal cells that were directly differentiated
from iPSCs14. Cells in the iPSC-LB efficiently differentiate into
functional hepatocytes at ectopic sites within the mouse in the
absence of any external signals, such as severe liver injury, suggest-
ing that spatiotemporal multicellular interactions, including the
endothelial populations, have important roles in the successful
maturation of the derived hepatocytes.
Generation of a vascularized and functional human
liver from an iPSC-derived organ bud transplant
Takanori Takebe1–3, Ran-Ran Zhang1,5, Hiroyuki Koike1,5, Masaki Kimura1,5, Emi Yoshizawa1,5,
Masahiro Enomura1, Naoto Koike1,4, Keisuke Sekine1 & Hideki Taniguchi1,2
1Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Japan. 2Advanced Medical Research Center, Yokohama City
University, Yokohama, Japan. 3Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Kawaguchi, Japan.
4Department of Surgery, Seirei Sakura Citizen Hospital, Ebaradai, Sakura, Japan. 5These authors contributed equally to this work. Correspondence should be addressed to
T.T. (ttakebe@yokohama-cu.ac.jp) or H.T. (rtanigu@yokohama-cu.ac.jp).
Published online 23 January 2014; doi:10.1038/nprot.2014.020
Generation of functional and vascularized organs from human induced pluripotent stem cells (iPSCs) will facilitate our
understanding of human developmental biology and disease modeling, hopefully offering a drug-screening platform and providing
novel therapies against end-stage organ failure. Here we describe a protocol for the in vitro generation of a 3D liver bud from
human iPSC cultures and the monitoring of further hepatic maturation after transplantation at various ectopic sites. iPSC-derived
specified hepatic cells are dissociated and suspended with endothelial cells and mesenchymal stem cells. These mixed cells
are then plated onto a presolidified matrix, and they form a 3D spherical tissue mass termed a liver bud (iPSC-LB) in 1–2 d.
To facilitate additional maturation, 4-d-old iPSC-LBs are transplanted in the immunodeficient mouse. Live imaging has identified
functional blood perfusion into the preformed human vascular networks. Functional analyses show the appearance of multiple
hepatic functions in a chronological manner in vivo.
© 2014 Nature America, Inc. All rights reserved.
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NATURE PROTOCOLS | VOL.9 NO.2 | 2014 | 397
Advantages and disadvantages of the
present protocol
Alternative methods to create a whole
organ are based on the concept of tissue
engineering, as initially stated by Langer
and Vacanti15. Tissue engineering generally
relies on the use of a combination of cells,
factors and scaffolds. Recent advances in
whole-organ decellularization techniques
have enabled the fabrication of promis-
ing scaffolds for the engineering of new
organs, including the heart, liver, lung and
kidney16,17. However, these tissue engineering studies require
several crucial improvements to be translated to cell derivatives of
human pluripotent stem cells, because matrices, spatial relation-
ships and molecular stimuli intercommunicating across various
cellular types shift rapidly from pluripotent states during the early
stages of embryonic development. The complexity and fluidity of
organogenesis is difficult to mimic by conventional tissue engineer-
ing approaches such as the use of adult organ–derived decellularized
matrices. These matrices or artificial scaffolds are not fluidic but
static, and thus they markedly limit the natural changes in cellular
or molecular interactions in a spatiotemporal manner18. In contrast,
our approach essentially reconstitutes the multiple cellular interac-
tions that are important for organogenesis, which enables the cells to
self-organize into a 3D tissue presumably under natural spatiotem-
poral orientation. Upon transplantation, the cells undergo efficient
differentiation into target cell types stimulated in a chronological
manner by experiencing organogenetic spatiotemporal changes with
supporting stromal cells, mechanical load and growth stimuli cor-
responding to the appropriate developmental stages.
In this article, we describe a detailed protocol for the genera-
tion of liver buds from human iPSCs (Fig. 1a) and their trans-
plantation at various ectopic sites (Fig. 1b). Establishing a system
for the de novo generation of vascularized and functional organs
from iPSCs should provide a unique opportunity to study human
developmental biology and disease modeling, and it may supply
a drug-screening platform. Given the unsatisfactory clinical out-
comes of the cell-based therapies that are the current main target
of stem cell therapy, our proof of concept, i.e., organ bud trans-
plantation, could revolutionize generative medicine for treating
end-stage organ failure.
Experimental design
Preparation of human iPSC–derived hepatic endoderm cells
(iPSC-HEs). We primarily use the TkDA3 human iPSC line
reprogrammed from dermal fibroblasts, which is maintained on
mitotically inactivated primary embryonic feeder cells (mouse
embryonic fibroblasts (MEFs)) before differentiation culture.
For live imaging analysis in vitro, GFP knock-in reporters for the
expression of adeno-associated virus integration site 1 (AAVS1::
GFP) are used. To derive iPSC-HEs, we use the two-stage dif-
ferentiation methods that have been previously described6.
In the first stage, human iPSCs are seeded on a Matrigel-coated dish
and the medium is changed to RPMI-1640 with 1% (vol/vol) B27
containing human activin A but not insulin (100 ng ml−1) for
6 d. Immunocytochemistry using antibodies to detect proteins
expressed in the definitive endoderm shows that more than 90%
of cells expressed forkhead box A2 (FOXA2) and SRY-box 17
(SOX17). For hepatic specification, human iPSC-derived endo-
dermal cells are treated further with RPMI-1640 with 1% B27,
human basic fibroblast growth factor (FGF; 10 ng ml1) and
human bone morphogenetic protein 4 (BMP4; 20 ng ml−1) for
3 d. We primarily assess hepatic cell specification by hepatocyte
nuclear factor 4α (HNF4A) expression, because HNF4A expres-
sion is restricted to the nascent hepatic cells that are formed dur-
ing the hepatic specification stages of development (Fig. 2a,b).
Note that before generating iPSC-LB, iPSC-HEs have not yet
expressed α-fetoprotein (AFP), which indicates that the specified
cells have been committed to a hepatoblast fate. We highly recom-
mend that the hepatic specification of human iPSCs be examined
at each time point of differentiation culture by immunostaining
and gene expression studies in addition to careful microscopic
observation, because successful differentiation in this step is cru-
cial for in vivo functional maturation.
Preparation of human endothelial and mesenchymal cells
We use human umbilical vein endothelial cells (HUVECs) and
human bone marrow–derived mesenchymal stem cells (hMSCs
(Fig. 2c,d). Special care should be taken to avoid overconfluency
of HUVECs and MSCs in the maintenance culture, because this
outcome could lead to the undesirable vascularization of the
transplants. For live imaging experiments, infect cells with retro-
viruses expressing EGFP or kusabira orange (KO1) as described
previously19. In brief, transfect the retrovirus vector pGCDNsam
IRES-EGFP or -KO1 into 293gp and 293gpg packaging cells
MSCs
6 d
+ActivinA
3 d
+bFGF
+BMP4
HUVECs
iPSC-HEs
EGM
MSC GM
MSCsHUVECs
Human iPSCs
Cell number
2 × 106 cells in total; 24 wells
iPSC-HEs: HUVECs: MSCs = 10:7:2
a b
Manipulation
Matrigel pre solidification
Pick up and transplantation
3–6 d
Proximal mesentery
Subrenal capsule
Distal mesentery
Cranium
Transplantation into
immunodeficient mice
Medium
HCM without EGF diluted with EGM (1:1)
Figure 1 | Protocol for generating a vascularized
and functional human liver from iPSC–derived
organ bud transplants. (a) Timetable for growing
human liver buds in vitro from human iPSCs
by recapitulating ontogenetic multicellular
interactions. (b) Schematic diagram of our
transplantation strategy. Self-organized 3D
human iPSC-LBs are transplanted at various sites,
including the cranium, subrenal capsule and two
mesenteric sites.
© 2014 Nature America, Inc. All rights reserved.
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398 | VOL.9 NO.2 | 2014 | NATURE PROTOCOLS
(in which viral particle production is induced by using a tetracycline-
inducible system). Use the culture supernatants of the retrovirus-
infected cells for infection. KO1 displays a major absorption
wavelength maximum at 548 nm with a slight shoulder at 515 nm,
and it emits a bright orange fluorescence with a peak at 561 nm.
Human iPSC-LB formation. To mimic early organogenesis
in vitro, hepatic endoderm cells should be cultured with endothe-
lial and mesenchymal lineages on solidified gels comprising base-
ment membrane matrix mixtures. For the liver bud culture, we
use 500 ml of hepatocyte culture medium (HCM)epidermal
growth factor (EGF), 25 ml of FBS CELLect Gold, 100 nM
dexamethasone, 20 ng ml−1 oncostatin M and 10 ng ml−1 hepa-
tocyte growth factor (HGF), which is mixed with an equal amount
of endothelial growth medium (EGM) (HCM/EGM). Cells initi-
ate 3D organization within a few hours and finally form a 3D liver
bud within 48 h (Fig. 2e,f). In our previous reports14,20, we vali-
dated the various solidified gels and concentrations and showed
that Matrigel was the most efficient for iPSC-LB formation and
led to better transplant outcomes in vivo. Human iPSC-LBs gener-
ated in a 24-well plate are visible by macroscopic observation after
1-d culture (Fig. 2g). Analysis with a fluorescence microscope will
demonstrate the rigorous endothelial sprouting inside the iPSC-
derived liver bud after 6 d of culture (Fig. 2h).
Once human iPSC-LBs are generated, the tissues can be manip-
ulated with any preferable instruments, because they are mechani-
cally stable. For transplant use, we routinely collect the liver bud
with a spatula or a wide-opening P-1000 pipette. In this study, we
describe the detailed transplant protocols at multiple potential
ectopic sites (Figs. 35).
Transplantation under cranial window The cranial window
method is a useful tool for analyzing live brain physiology and
angiogenesis or vasculogenesis, but it has rarely been evaluated for
assessing endodermal organ development. In our recent report20,
we demonstrated the feasibility of using the cranial window to
0 h 2 h 4 h
10 h 20 h6 h
e
0
100
200
CXCR4
iPSCs
iPSC-DEs
iPSC-HEs
iPSCs
iPSC-DEs
iPSC-HEs
0
200
400
600
CER1
iPSCs
iPSC-DEs
iPSC-HEs
iPSCs
iPSC-DEs
iPSC-HEs
0
100
200
300
FOXA2
0
20
40
60 HNF4A
b
0
0.4
0.8
1.2
iPSCs
iPSC-DEs
iPSC-HEs
iPSCs
iPSC-DEs
iPSC-HEs
NANOG
0
0.4
0.8
1.2 OCT4
Relative expressions (fold change)
f
0 h 2 h 4 h
10 h 20 h6 h
iPSC-HEs HUVECs MSCs
MSCs
aiPSC-HEsiPSC-DEsiPSCs
c d MSCsHUVECs
g
iPSC-HEs
only
iPSC-HEs
HUVECs MSCs
HUVECs
iPSC-HEs
h
Figure 2 | Generation of 3D liver buds from iPSCs in vitro by recapitulating early organogenetic interactions. (a) Hepatic specification of human iPSCs by
directed differentiation. Scale bars, 200 µm. (b) Quantitative PCR analysis of various marker genes for pluripotency, definitive endoderm and hepatic endoderm
in human iPSC-HEs at day 9 of culture. Methods used were given in ref. 14. Control samples show the expression of human iPSC and iPSC-definitive endoderm
cells (iPSC-DEs). Results represent the mean ± s.d., n = 3. (c,d) Representative microphotographs showing the typical morphology of HUVECs (c) and MSCs (d).
Scale bars, 200 µm. (e,f) Self-organization of 3D human iPSC-LBs in cocultures of human iPSC-HEs with HUVECs and human MSCs (Supplementary Video 1).
The time-lapse fluorescence imaging of human iPSC-HEs is shown in f. Scale bars, 5 mm (e), 500 µm (f). Green, human iPSC-HEs; red, HUVECs; blue, human
MSCs. (g) Gross observation of human iPSC-LBs (top panel) and conventional 2D cultures (bottom panel). Scale bar, 5 mm. (h) Presence of human iPSC-HEs and
nascent endothelial networks inside human iPSC-LBs after day 3 of culture. Human iPSC-HEs are green, HUVECs are red and MSCs are blue. Scale bar, 250 µm.
© 2014 Nature America, Inc. All rights reserved.
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NATURE PROTOCOLS | VOL.9 NO.2 | 2014 | 399
study liver cell maturation and vascular morphology and func-
tion using transplants of embryonic day (E)13.5 mouse fetal liver
cells, human fetal liver cells and human iPSC-hepatic endoderm
cells. This transplantation approach provided a unique intravital
monitoring system for evaluating human iPSC–derived organ
maturation and differentiation throughout organogenesis, and
it allowed us to dissect the previously uncharacterized roles of
stromal cell types in human organ development.
After removing the preformed cranial window of immuno-
deficient mice, a human iPSC-LB (4–6 mm) is placed on the
brain surface and then sealed with an 8-mm cover glass that is
adhered to the bone. Gross observation by live imaging analysis
shows the signs of blood perfusion inside the human vessels of
transplanted human iPSC-LBs as early as 48–72 h (Fig. 4a,b).
These vessels become progressively more mature throughout the
course of transplantation (Fig. 4c). Intravital confocal micro-
scope analyses show that fluorescent human endothelial cells
form tubular vessel-like structures that contain blood flow, as
visualized by fluorescence-conjugated dextran infusion via the tail
vein (Fig. 4d,e). By combining different fluorescently labeled cells,
we can identify the spatiotemporal localization and morphology
of each engrafted cell type at the appropriate timing (Fig. 4f,g).
For instance, KO1-labeled human MSCs exist with a perivascular
localization at 14 d, indicating pericyte differentiation for the sta-
bilization of human vessels (Fig. 4f). At this stage, we can identify
the direct connections between human blood vessels and host
vessels within the transplant by infusing fluorescein-conjugated
dextran and a mouse CD31 antibody (Fig. 4h).
Other potential ectopic sites for trans-
plantation Aiming at future clinical
applications, the cranial window model
is not an efficient method for organ bud
transplantation because of its reduced
u v w x y
Distal
mesentery Cranium
a b c d e
f g h i j
k l m n o
p q r s t
Proximal
mesentery
Subrenal
capsule
Figure 3 | Ectopic transplantation of human
iPSC-derived liver buds at various sites.
(aj) Procedures for the cranial window
preparation (ae) and transplantation of human
iPSC-LBs (fj) after 1 week. See PROCEDURE
(Step 40A) for details of what is shown in each
individual panel and Supplementary Videos 2
and 3. (ko) Procedures for subrenal capsule
transplantation. See the PROCEDURE (Step 40B)
for details of what is shown in each individual
panel and Supplementary Video 4.
(pt) Procedures for distal mesenteric
transplantation. See PROCEDURE (Step 40C) for
details of what is shown in each individual panel
and Supplementary Video 5. (uy) Procedures
for mesenteric transplantation proximal to the
liver. The periportal capsule should be carefully
removed for the transplants to improve the
functional outcomes after transplantation.
See PROCEDURE (Step 40D) for details of
what is shown in each individual panel and
Supplementary Video 6. The white dashed line
indicates the transplanted human iPSC-LBs.
0 d 3 d 14 d
iPSC-HEs HUVECs MSCs Dextran iPSC-HEs HUVECs MSCs Dextran iPSC-HEs HUVEC MSCs Dextran
iPSC-HEs HUVECs MSCs mCD31 Dextran
a cb
d fe g
h
Figure 4 | Intravital visualization of vascularized
and functional human liver formation from
iPSC-derived liver buds inside the cranium.
(ac) Macroscopic observation of transplanted
human iPSC-LBs, showing the perfusion of
human blood vessels (c, right). Scale bar,
2 mm. (d,e) Functional human blood vessel
formation in vivo. Green, dextran; red, HUVECs.
(f,g) Localization of human MSCs (green,
dextran; red, human MSCs) or human iPSC-derived
cells (green, dextran; red, human MSCs) at day
15. Scale bars, 250 µm. The dashed line indicates
the edge of transplanted human iPSC-LBs.
(h) Visualization of the connections between
HUVECs (green) and host vessels (red) containing
functional blood flow (blue). Scale bars, 50 µm.
Black, nonlabeled cells in all panels.
© 2014 Nature America, Inc. All rights reserved.
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400 | VOL.9 NO.2 | 2014 | NATURE PROTOCOLS
scalability and high level of invasiveness. Therefore, we sought
to examine the feasibility of three other ectopic transplanta-
tion models: the subrenal capsule (Fig. 3k–o) and mesenteric
sites, including locations that are distal (Fig. 3p–t) and proximal
(adjacent site to portal vein) (Fig. 3u–y) to the recipient liver. The
functional output of a human liver can be evaluated by multiple
quantitative analyses including FACS, protein production and
drug metabolism. For example, human-specific albumin produc-
tion measured by an ELISA (Bethyl Laboratories) can be routinely
monitored by using blood serum drawn from transplanted mice,
and this assay suggests that mesenteric transplantation at a site
proximal to the liver is the most efficient method for hepatocyte
engraftment and terminal differentiation (Fig. 5).
0
200
400
600
800
1,000
1,200
Cranium
iPSC-LB ×3
Subrenal
capsule
iPSC-LB ×8
Distal
mesentery
iPSC-LB ×8
Proximal
mesentery
iPSC-LB ×8
Figure 5 | Functional characterization of human iPSC-LB transplants
at various ectopic sites based on in vivo albumin (ALB) production.
Functional evaluation using ELISA was carried out as described in ref. 14.
This demonstrates levels of human serum ALB (ng ml−1) present 30 d after
transplant at various ectopic sites (cranium, kidney subrenal capsule, distal
mesentery and proximal mesentery). Numbers after the multiplication symbol
(×) indicate the number of transplanted iPSC-LBs. Error bars are means ± s.d.,
n = 4 (ref. 14).
MATERIALS
REAGENTS
iPSCs (we use TkDA3, kindly provided by K. Eto and H. Nakauchi)
CRITICAL We have used various other iPSC cell lines for self-organization
experiments; however, we strongly recommend that intensive screening be
carried out initially to determine the most efficient line to use. The
optimum line should be the one that produces the most albumin in vivo
after transplantation, as this indicates the best final functional output.
MEFs from E13.5 embryos ! CAUTION Experiments involving rodents must
conform to all relevant institutional and governmental regulations.
HUVECs (Lonza, cat. no. 191027)
hMSCs (Lonza, cat. no. PT-2501)
Human 293-derived retroviral packaging cell line (293gp and 293gpg lines)
Ca2+- and Mg2+-free Dulbecco’s PBS (D-PBS(−); Wako, cat. no. 045-29795)
PBS(−) powder, 1× (Wako, cat. no. 162-19321)
DMEM, high glucose (Wako, cat. no. 043-30085)
D-MEM/F-12, 1:1 (Gibco, cat. no. 11330-032)
RPMI-1640 (Wako, cat. no. 189-02025)
Mesenchymal stem cell growth medium (MSCGM ) BulletKit (Lonza,
cat. no. PT-3001)
EGM BulletKit, the basal medium is stable for 3 months at 4 °C (Lonza,
cat. no. CC-3124)
HCM BulletKit (Lonza, cat. no. CC-3198)
FBS, for mouse fibroblast culture (Biowest, cat. no. S1560-500)
FBS, CELLect GOLD, for hepatic differentiation; store it at −20 °C after
complement heat inactivation (MP Biomedicals, cat. no. 29169)
KnockOut serum replacement (KSR; Gibco, cat. no. 10828-028)
B-27 supplement (50×) containing insulin (Gibco, cat. no. 17504-044)
B-27 supplement minus insulin (50×) (B27insulin; Gibco, cat. no.
0050129SA)
Matrigel, matrix growth factor reduced (Matrigel GFR; BD, cat. no. 354230)
BD Matrigel matrix (BD Biosciences, cat. no. 356234)
Gelatin type B (Sigma, cat. no. G9391)
Rho-associated kinase (ROCK) inhibitor Y-27632 (Wako, cat. no. 253-00513)
Accutase (Funakoshi, cat. no. AT104)
Trypsin-EDTA, 0.05% (wt/vol); freeze aliquots at −20 °C for long-term
storage or thaw them and store them at 4 °C for a couple of months
(Gibco, cat. no. 15400-054)
Trypan blue stain (Gibco, cat. no. 15250-061)
GlutaMAX supplement (Gibco, cat. no. 35050-061)
Human activin A (R&D Systems, cat. no. 338-AC)
Human basic fibroblast growth factor (hbFGF; R&D Systems,
cat. no. 233-FB)
BMP4 (R&D Systems, cat. no. 314-BP)
Oncostatin M (OSM; R&D Systems, cat. no. 295-OM)
MEM non-essential amino acids (NEAA; 100× solution; Gibco,
cat. no. 11140-050)
HEPES (Wako, cat. no. 346-08235)
l-glutamine, 200 mM (Gibco, cat. no. 25030)
2-Mercaptoethanol (2-ME; 1,000× solution; cat. no. 21985-023)
! CAUTION 2-ME is toxic. When used, avoid inhalation and skin contact.
Penicillin-streptomycin (Gibco, cat. no. 15140122)
Dexamethasone (Sigma, cat. no. D2915)
Human recombinant HGF (Sigma, cat. no. H1404)
DMSO (Mallinckrodt Baker, cat. no. 7033)
Cell Banker1 (Wako, cat. no. 630-01601)
Liquid nitrogen
Immunodeficient (NOD/SCID) mice (we obtain our mice from Sankyo
Lab) ! CAUTION Experiments involving live rodents must conform to all
relevant institutional and governmental regulations.
Tetramethylrhodamine-conjugated dextran, molecular weight (MW)
2,000,000 Da (Invitrogen, cat. no. D-7139)
Fluorescein isothiocyanate–conjugated dextran, MW 2,000,000 Da
(Invitrogen, cat. no. D-7137)
Texas Red–conjugated dextran, MW 70,000, neutral, (Invitrogen,
cat. no. D-1830)
Alexa Fluor 647 mouse anti-human CD31 (BioLegend, cat. no. 303112)
Alexa Fluor 647 rat anti-mouse CD31 (BioLegend, cat. no. 102516)
Ethanol (Wako, cat. no. 059-07895) ! CAUTION Ethanol is flammable.
Avoid exposure to ignition.
Distilled water
Normal saline (Otsuka Pharmaceutical)
Fibrin sealant (Bolheal; Kaketsuken)
Isoflurane (Mylan)
Cyanoacrylate glue (Krazy glue; Toagosei)
Dental cement (Coe tray plastic; GC America, cat. no. 240012)
Ketamine (Mediceo)
Xylazine (Webster)
Iodine tincture (Toho Pharmaceutical)
EQUIPMENT
CO2 incubators, 2.5% and 5% (Panasonic)
Centrifuge (Hitachi Koki)
Tissue culture dish, 100 mm (BD, cat. no. 353003) CRITICAL The brand
of culture dish may have an effect on the quality of the human iPSC culture
and the efficacy of cell attachment to gelatin.
© 2014 Nature America, Inc. All rights reserved.
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NATURE PROTOCOLS | VOL.9 NO.2 | 2014 | 401
Tissue culture dish, 6 cm (BD, cat. no. 353002)
Tissue culture six-well plate (BD, cat. no. 353046)
Cell culture 24-well plate (BD, cat. no. 353047)
High-clarity polypropylene conical tube, 15 ml (BD, cat. no. 352096)
Polypropylene conical tube, 50 ml (BD, cat. no. 352070)
Water bath (Fisher Scientific)
Milli-Q water purification system (Millipore)
Microcentrifuge tube, 1.5 ml, autoclavable (Fisher Scientific,
cat. no. 05-408-129)
Pipette-aid, filler/dispensers (Drummond)
Liquid waste disposal system for aspiration
Glass Pasteur pipette (IWAKI, cat. no. IK-PAS-5P-B)
Hemocytometer, Reichert Bright-Line counting chamber (Fisher Scientific,
cat. no. 02-671-6)
Sterile biosafety cabinet (Panasonic)
Phase contrast microscope (Olympus)
Fluorescence microscope (Keyence model BZ-9000)
Leica TCS SP5 confocal microscope (Leica Microsystems)
MetaMorph angiogenesis module software (Molecular Devices)
Anesthetic vaporizer (Shinano Manufacturing)
Spatula (Fine Science Tools)
Heating bed (As one)
Toothpicks (Yanagi Products)
Needles (Terumo)
Square coverslips (Matsunami Glass, cat. no. CO25601)
Circular coverslips (Matsunami Glass)
High-speed micro-drill (Fine Science Tools)
Gelatin sponge (Spongel; Astellas)
Stereoscopic microscope (Leica)
Sutures (Natsume Seisakusho)
Drape (Konya paper)
Scotch tape (Nichiban)
REAGENT SETUP
FBS Thaw the FBS. To inactivate the complement, heat the closed bottle for
30 min at 56 °C. Later, divide the FBS into aliquots and store them at −20 °C
until use. These aliquots can be stored for up to 12 months.
bFGF stock solution Reconstitute 50 µg of bFGF powder with 500 µl of
deionized water to a concentration of 100 ng µl−1; next, divide the solution
(50 µl per tube) into aliquots and store them at −20 °C until use. These
aliquots can be stored for up to 3 months.
bFGF working solution Retrieve 50 µl of bFGF stock solution from storage
at −20 °C. Dilute it with 950 µl of 0.1% (wt/vol) BSA per 5 mM Tris-HCl to a
final concentration of 5 µg ml−1. Store this solution at 4 °C. This solution can
be stored for up to 2 weeks.
Dexamethasone working solution Dissolve water-soluble dexamethasone
powder (65 mg) in 16.6 ml of cell culture–grade water, filter-sterilize the
solution and store it frozen in small aliquots. This stock solution is 10 mM;
dilute the stock solution to 1 µM before use. Store this solution at −20 °C.
This solution can be stored for up to 2 months.
Oncostatin M working solution Dissolve a total of 50 µg of human
oncostatin M powder in 500 µl of 0.1% (wt/vol) BSA/PBS to a concentration
of 100 ng µl−1 and store the 50-µl aliquots at −80 °C. Before use, thaw a stock
tube and add 200 µl of 0.1% (wt/vol) BSA/PBS to dilute it to 20 ng µl−1.
Further dilute the solution 1,000× to generate the working solution.
Store this solution at 4 °C. This solution can be stored for up to 2 weeks.
HGF working solution Dissolve human recombinant HGF powder in 0.1%
(wt/vol) BSA/PBS to a concentration of 5 µg ml−1; use a 250× dilution of this
solution to yield the final concentration of 20 ng ml−1. Store this solution at
−20 °C. This solution can be stored for up to 2 months.
ROCK inhibitor Add 1,500 µl of cell culture–grade water to 5 mg of ROCK
inhibitor to obtain a 10 mM stock solution and store the aliquots at −30 °C.
The final working concentration is 10 nM. This solution can be stored for up
to 2 months.
BMP4 Dissolve 500 µg of BMP4 in 5 ml of 4 mM HCl and 0.1% (wt/vol)
BSA/PBS to yield a 100 ng µl−1 stock solution and store the aliquots
at −30 °C. This solution can be stored for up to 2 months. Before use,
thaw a tube of stock solution and dilute 5 × 103 to obtain the final
working solution.
Activin A Dissolve a total of 25 µg of human activin A in 400 µl of 0.1%
(wt/vol) BSA/PBS to yield a 100 ng µl−1 stock solution and dilute it to a
final concentration of 100 ng ml−1. Store the stock solution at −20 °C.
This solution can be stored for up to 2 months. CRITICAL The 0.1%
(wt/vol) BSA/PBS used above should be sterilized. All the growth factors and
cytokines used in this experiment should be manipulated according to the
manufacturer’s procedures.
Gelatin (0.1% (wt/vol)) solution for coating the tissue culture plates
(500 ml) Combine 0.5 g of gelatin with 500 ml of deionized water and then
dissolve it. Sterilize the solution in an autoclave at 120 °C for 20 min,
remembering to cool it to room temperature (18–28 °C) before use.
This solution can be stored at room temperature for up to 2 months.
Matrigel (1:30) for plate coating (30 ml) Add 29 ml of prechilled, pure
DMEM (no serum, no additive and no antibiotic) to a 50-ml BD Falcon
tube on ice, retrieve 1 ml of Matrigel from storage at 4 °C and resuspend the
Matrigel in cold medium. To prevent solidification, avoid placing the solution
at room temperature.
Medium for MEF culture (500 ml) Mix 500 ml of DMEM, 50 ml of FBS and
5 ml of penicillin-streptomycin. This medium can be stored at 4 °C for up to
a month.
Medium for human iPSC maintenance culture on MEF feeder cells
(iPSC-M; 500 ml) Combine 500 ml of DMEM-F12, 125 ml of KSR, 5 ml of
NEAA, 6.25 ml of GlutaMAX and 1,200 µl of 2-ME. This medium can be
stored at 4 °C for up to 2 weeks. Prewarm the medium to 37 °C before use.
Medium for definitive endoderm differentiation culture (100 ml) Mix
100 ml of RPMI-1640, 1 ml of penicillin-streptomycin and 1 ml of insulin-
reduced B27 supplement. This mixture can be stored at 4 °C for up to
1 month and should be prewarmed to 37 °C before use.
Medium for hepatic endoderm differentiation culture (100 ml) Mix 100 ml
of RPMI-1640, 1 ml of penicillin-streptomycin and 1 ml of B27 supplement
(not insulin reduced). This mixture can be stored at 4 °C for up to 1 month
and should be prewarmed to 37 °C before use.
Dissociation solution for human iPSC passage (50 ml) Mix 5 ml of 2.5%
(wt/vol) trypsin, 10 ml of KSR, 500 µl of 100 mM CaCl2/PBS and 34.5 ml of
PBS. Divide the solution into 10-ml aliquots in 15-ml BD Falcon tubes and
store the stock solution at −20 °C for up to 2 months. Thaw the solution at
4 °C and use it within 1 week of thawing.
Medium for endothelial cell maintenance (EGM) Thaw the reagents
enclosed in the EGM BulletKit: bovine brain extract (BBE), human
epidermal growth factor (hEGF), hydrocortisone, FBS, GA-1000 and ascorbic
acid. Add the supplementary reagents included in the BulletKit to 500 ml of
endothelial basal medium (EBM) to produce complete EGM. This medium
can be stored at 4 °C for up to 1 month. CRITICAL The culture medium
is referred to as EBM before the addition of the supplement; afterward,
the medium is referred to as EGM.
Medium for mesenchymal stem cell maintenance (MSCGM) Thaw the
reagents enclosed in the MSCGM BulletKit: mesenchymal cell growth
supplement (MCGS), l-glutamine and GA-1000. Add the supplementary
reagents included in the BulletKit to 440 ml of mesenchymal stem cell basal
medium (MSCBM) to produce complete MSCGM. This medium can be
stored at 4 °C for up to 1 month. CRITICAL The culture medium is referred
to as MSCBM before the addition of the supplement; afterward, the medium
is referred to as MSCGM.
Medium for the iPSC-LB self-organization culture (HCMEGF) Thaw
the reagents enclosed in the HCM BulletKit: transferrin, hydrocortisone,
BSA–fatty acid free (BSA-FAF), ascorbic acid, insulin and GA-1000. Add
the supplementary reagents, omitting hEGF, from the BulletKit to 500 ml
of hepatocyte basal medium (HBM) to produce complete HCM without
hEGF (HCMEGF). This medium can be stored at 4 °C for up to 1 month.
CRITICAL The BulletKit comes with hEGF, but this reagent is not added to
the medium.
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402 | VOL.9 NO.2 | 2014 | NATURE PROTOCOLS
PROCEDURE
Maintenance of human iPSCs
1| Plating MEFs on a coated dish to generate a feeder layer for human iPSCs. Coat a 100-mm dish with an appropriate
volume of 0.1% (wt/vol) gelatin; it is necessary to use treated polystyrene cell culture plates. For a 100-mm dish, use
5 ml. For a 60-mm dish, use 2 ml. Shake the plate vigorously after adding the coating solution to evenly coat the surface.
2| Incubate the plates at room temperature for ~15 min.
3| After incubation, remove the coating solution and add the MEF (mitomycin C–treated) suspension in MEF culture
medium. Optimized MEF cell numbers are 1.8 × 106 for a 100-mm dish and 0.6 × 106 for a 60-mm dish. It is important to
seed the feeder cells at the appropriate density to avoid human iPSC differentiation. MEFs seeded at this density can usually
be used for human iPSC culture 1–2 d after seeding.
4| Incubate MEFs at 37 °C and 5% CO2 overnight. Check the cell density until they reach 70–80% confluency (this should
correspond to 0.5–1 × 106 cells per 100-mm plate).
5| Maintenance of human iPSCs on the feeder layer. Passage the iPSCs onto fresh MEF plates from Step 4 once the iPSCs
have reached 30% confluency (usually after 3–5 d). If 30% confluency has not been reached after 6 d of culture, passage the
cells anyway. Healthy human iPSCs should appear with clear colony edges and high cell density in the center of the colony.
The suitable colony size for passaging should be between 1 and 2 mm in diameter (Fig. 2a). When desired, proceed to the
next step.
6| Take human iPSCs at 30% confluency in a 100-mm dish, aspirate the medium and wash it with 5 ml of PBS. Remove the
PBS. Add 1.5 ml of dissociation solution and ensure that the solution covers all the cells.
7| Incubate the cells at 37 °C, 2.5% CO2, for 7 min; observe the culture under a microscope to confirm that the colony
starts to detach.
8| Discard the dissociation solution by aspiration, wash it with 5 ml of human iPSC-M and aspirate and discard the
wash medium.
9| Pipette the colony into fragments sized 100–200 µm, and then use a P-1000 pipette to place each fragment in 1 ml of
human iPSC-M, to a total volume of 10 ml.
CRITICAL STEP Avoid completely dissociating the colony, because human iPSCs cannot survive as single cells.
10| Take a fresh MEF-containing dish at a cell density of 70–80% confluency (from Step 4), discard the MEF medium,
wash it with PBS and aspirate and discard the PBS.
11| Add 9 ml of fresh iPSC-M plus 9 µl of bFGF working solution.
? TROUBLESHOOTING
12| Plate the medium (~1 ml) containing the dissected colonies from Step 9 into the dish described in Step 11.
? TROUBLESHOOTING
13| Culture cells with daily medium change for 3–5 d at 37 °C with 2.5% CO2.
Initiation of human iPSC differentiation
14| Check the morphology of the cells. If the human iPSC morphology shows that unhealthy or differentiated cells cover
over 20% of the dish, the cells should not be used. The optimized cell density for transferring cells onto a Matrigel-coated
dish should be such that the cells are likely to reach 100% confluency after an overnight culture. When the cells are ready
for re-plating, proceed to the next step to coat the dishes with Matrigel ready for further culture.
15| Add 2 ml of Matrigel working solution to a 60-mm dish or 200 µl to each well of a 24-well plate. Incubate the culture
at room temperature for 2 h and then return the Matrigel solution to the former tube for reuse and add pure DMEM-F12 to
each dish.
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NATURE PROTOCOLS | VOL.9 NO.2 | 2014 | 403
16| Rinse the dish containing human iPSCs (from Step 14) with an appropriate volume of sterile Ca2+/Mg2+-free PBS and
discard it. For a 100-mm dish, wash with 5 ml of PBS.
17| Add Accutase to the dish (1.5 ml per 100-mm dish); incubate the culture at 37 °C and 2.5% CO2 for 4 min.
CRITICAL STEP Check cell conditions under a microscope. If the cells are lifting away and floating from the bottom of the
dish, immediately neutralize them with 2 ml of fresh pure DMEM-F12 without cytokines.
18| Collect the cell suspension into a new 50-ml conical tube and perform a second wash of the plate with an additional
10 ml of pure DMEM-F12. Transfer the second wash into the same conical tube.
19| Extract 10 µl of the above cell suspension for cell counting.
20| Centrifuge the remaining cell suspension to collect the cells at 90g for 5 min at 4 °C. Aspirate the supernatant and
suspend the cell pellet with an appropriate volume of fresh human iPSC-M plus 5 ng ml−1 of bFGF and 10 µM of ROCK inhibitor.
For example, mix 10 ml of human iPSC-M with 10 µl of bFGF working solution and 10 µl of ROCK inhibitor working solution.
21| Aspirate the DMEM-F12 from the Matrigel-coated dish or plate (from Step 15), and then transfer the above cell
suspension onto the Matrigel-coated dish or plate at the proper cell density. For a 60-mm dish, use 1.5 × 106 cells per 4 ml
of medium. For a 24-well plate, use 1.5 × 105 cells per 0.5 ml of medium.
22| Incubate the culture at 37 °C and 5% CO2 overnight.
? TROUBLESHOOTING
23| On the next day (day 1 after plating), aspirate the medium, wash it with sterile PBS and remove the PBS. Replace it
with RPMI/B27insulin medium with 100 ng ml−1 of activin A added.
24| Continue to culture at 37 °C and 5% CO2 for an additional 5 d (days 2–6 after plating), with medium replacement every
2 d (Fig. 2a).
CRITICAL STEP The medium used at this stage should lead to the cell morphology changing in a similar way to the
definitive differentiation of endoderm cells; after 6 d of culture, over 80% of cells should express the endoderm marker
FOXA2 (Fig. 2b).
? TROUBLESHOOTING
25| On day 7 after plating, replace the medium with RPMI/B27 medium supplemented with 10 ng ml−1 bFGF and 20 ng ml−1
BMP4. Culture it for another 3 d, with medium changes every 2 d (Fig. 2a).
CRITICAL STEP Successful differentiation must be confirmed by demonstrating that HNF4A expression is initiated and
FOXA2 expression is maintained, whereas pluripotency marker expressions such as nanog homeobox (NANOG) are significantly
downregulated (Fig. 2b).
Self-organization of human iPSC-LBs in cocultures of iPSC-HEs with HUVECs and human MSCs
26| Preparation for cocultures. Mix 500 ml of HCMEGF, 25 ml of FBS CELLect Gold, 100 nM dexamethasone, 20 ng ml−1
oncostatin M and 10 ng ml−1 HGF. Next, combine equal amounts of this medium and EGM (HCM/EGM).
27| Retrieve the BD Matrigel solution from storage at 4 °C and mix it in an equal volume of EGM on ice (Matrigel:
EGM = 1:1). Add 400 µl of the Matrigel/EGM mixture to a 24-well plate and incubate the plate at 37 °C in a 5% CO2
incubator for at least 20 min.
CRITICAL STEP The Matrigel used in this step is not growth factor reduced, and EGM supplementation is important for
enhancing the survival of endothelial cells.
Collection of human iPSC-HEs
28| To initiate the self-organization of human iPSC-LBs, collect HUVECs and hMSCs from plates, as described in Boxes 1
and 2. Concurrently, collect iPSC-HE as described in the following steps.
CRITICAL STEP The work of collection of each type of cell (Boxes 1 and 2 and Steps 25–33) should be divided among
multiple experimenters and should be completed at approximately the same time to improve the cell survival rate.
CRITICAL STEP A total of 2.0–3.0 × 106 HUVECs should be obtained from an 80% confluent 100-mm dish. Whereas
1.0–1.5 × 106 hMSCs should be obtained from an 80% confluent 100-mm dish.
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404 | VOL.9 NO.2 | 2014 | NATURE PROTOCOLS
29| Aspirate the medium and then wash the iPSC-HEs with 1 volume of PBS(−). Minimal volumes will depend on the plate
used; however, we recommend adding 2 ml per 60-mm dish.
30| Aspirate the PBS(−) and replace it with a minimal volume of trypsin-EDTA. Incubate the cells at 37 °C until most of the
cells have detached from the bottom of the plate (~5 min). Once the cells have detached, add ~2–3 volumes of MEF medium
(10% FBS containing DMEM) to the dish.
31| Transfer the cells to a 15-ml tube. Wash the cells by centrifuging at 150g for 5 min at 4 °C; remove the supernatant and
resuspend the pellet in HCM/EGM. The minimal volume of HCM/EGM will depend on the number of dishes (0.25–0.5 ml per
60-mm dish).
32| Mix 50 µl of the cell suspension and 50 µl of trypan blue stain and count the number of cells by using a cell counter.
CRITICAL STEP A total of 0.5–1.0 × 106 human iPSC-HEs should be obtained from a 60-mm dish.
33| Adjust the concentration to 1 × 106 cells per ml with HCM/EGM.
? TROUBLESHOOTING
Cocultures of human iPSC-HEs with HUVECs and hMSCs
34| Take the Matrigel-coated plate made in Steps 26 and 27 from the incubator.
CRITICAL STEP Do not wash or aspirate the Matrigel bed.
35| Combine 1 ml each of the suspensions of human iPSC-HEs (from Step 33), HUVECs (derived as described in Box 1) and
hMSCs (derived as described in Box 2) into one 15-ml tube.
CRITICAL STEP The ratio of each type of cell is human iPSC-HEs:HUVECs:hMSCs = 10:7:2.
36| Centrifuge the mixture at 160g for 5 min at 4 °C and carefully remove as much of the supernatant as possible <20 µl of
supernatant remaining). Resuspend the cells in 500 µl of HCM/EGM with a P-1000 pipette.
37| Seed the cell suspension onto the Matrigel bed with a P-1000 pipette.
Box 1 | Maintenance, passaging and collection of HUVECs TIMING ~4 d
1. Passage. Aspirate all of the spent medium from an 80% confluent 100-mm dish of HUVECs and then add 5 ml of PBS(−). Remove
the PBS(−), add 1 ml of trypsin-EDTA and incubate the mixture at 37 °C in a CO2 incubator until the cells are less than 90% detached
(2–6 min).
2. Add 2 ml of EGM to the dish and transfer the detached cell suspension to a sterile 15-ml centrifuge tube.
3. To collect the remaining cells in the dish, add 2 ml of EGM to the dish and transfer the cell suspension into the 15-ml centrifuge
tube.
4. Centrifuge the 15-ml tube at 150g at 4 °C for 5 min.
5. Aspirate the medium gently without disturbing the pellet, and resuspend the cells in 2 ml of EGM.
6. Mix 50 µl of the cell suspension and 50 µl of trypan blue stain and count the number of cells by using a cell counter.
7. Adjust the concentration to 40,000 cells per ml with EGM
8. Seed 10 ml of the cell suspension onto a 100-mm dish (4.0 × 105 cells per 100-ml dish), and then incubate the dish at 37 °C in a
humidified atmosphere containing 5% CO2.
9. Maintenance. Feed the HUVECs the day after seeding and every other day thereafter by replacing the old medium with 10 ml of
prewarmed EGM. After 2–3 d, the cells become 80% confluent (Fig. 2c). Passage the cells by using the same procedure described above
in steps 1–8.
10. Collection. Aspirate all of the spent medium from an 80% confluent 100-mm dish of HUVECs and add 5 ml of PBS(−). Remove the
PBS(−), add 1 ml of trypsin-EDTA and incubate the mixture at 37 °C in a CO2 incubator until the cells are <90% detached (2–6 min).
11. Once the cells have detached, add 2 ml of EGM to the dish.
12. Transfer the cells to a 15-ml tube. Wash the cells by centrifuging at 150g for 5 min at 4 °C, removing the supernatant and
resuspending the pellet in EGM. The minimal volume of EGM will depend on the number of dishes (1–2 ml per 100-mm dish).
13. Mix 50 µl of the cell suspension and 50 µl of trypan blue stain and count the number of cells with a cell counter.
14. Adjust the concentration to 700,000 cells per ml with EGM.
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NATURE PROTOCOLS | VOL.9 NO.2 | 2014 | 405
38| Add 1 ml of HCM/EGM to each well and incubate the plate at 37 °C in a humidified atmosphere of 5% CO2. Within 12 h,
the cells should start moving markedly, and they should form a 3D liver bud (Fig. 2eh). Feed the cocultures the day after
seeding and every other day thereafter by replacing the old medium with 1 ml of prewarmed HCM/EGM.
CRITICAL STEP The medium changes should be performed gently, without disturbing the LBs.
? TROUBLESHOOTING
39| If desired, monitor the cocultured cells in a 24-well plate under a stereoscopic or fluorescence microscope for time-lapse
analysis for up to 20 h (Fig. 2e,f). Figure 2g shows a generated iPSC-derived liver bud (Fig. 2g) (Supplementary Video 1).
Transplantation of in vitro–grown human iPSC-LBs
40| Transplant the 3-d-cultured iPSC-LBs (liver buds) into under a cranial window (option A, modified from method used in
Yang et al.21 and Xu et al.22; Supplementary Videos 2 and 3), under the kidney capsule (option B, modified from method
used in Zmuda et al.23, see also Supplementary Video 4), onto the distal mesentery (option C, Supplementary Video 5) or
onto the proximal mesentery (option D, Supplementary Video 6).
! CAUTION All surgical instruments must be sterilized.
(A) Transplant under a cranial window
(i) Preparation of the cranial window (Supplementary Video 2). Anesthetize an immunodeficient mouse by an i.p.
injection (0.2 ml per 20 g body weight) of 20 mg ml−1 ketamine and 3 mg ml−1 xylazine in 0.9% (wt/vol) NaCl.
(ii) Thoroughly shave the hair over most of the scalp with an electric shaver. Make a scalp incision by using sterile
microsurgical tools. The incision should extend approximately from the neck region to the frontal area.
(iii) Remove the soft tissue attached to the skull over the area to be imaged with a cotton swab. Use a high-speed
micro-drill to thin a circular region (typically ~7 mm in diameter) in the skull over the region of interest under a
dissection microscope (Fig. 3a).
(iv) Lift the island of bone within the drilled circle with a pair of sharp forceps, taking great care (Fig. 3b). Immediately
after removing the 7-mm-diameter region of the skull, bleeding above the dura may occur, presumably from small
blood vessels attached to the removed skull. Such bleeding should stop spontaneously within 10–20 s. Rinse the
cortex with normal saline once or twice if necessary.
(v) Remove the dura on the right and left sides of the brain without causing injury with a pair of sterile sharp tweezers.
At this step, make sure that there is absolutely no bleeding in the cortex (Fig. 3c).
(vi) Lower a circular coverslip (8 mm in diameter to cover the open skull (Fig. 3d).
(vii) Seal the edge of the optical window to the skull with a compound of cyanoacrylate glue and dental cement (Fig. 3e).
The compound will also cover the exposed skull.
Box 2 | Maintenance, passaging and collection of human MSCs TIMING ~7 d
1. Passaging. Aspirate all of the spent medium from an 80% confluent 100-mm dish of human MSCs and then add 5 ml of PBS(−).
Remove the PBS(−), add 1 ml of trypsin-EDTA and incubate the dish at 37 °C in a CO2 incubator until the cells are <90%
detached (2–6 min).
2. Add 2 ml of MSCGM to the dish and transfer the detached cell suspension to a sterile 15-ml centrifuge tube.
3. To collect the remaining cells in the dish, add 2 ml of MSCGM to the dish and transfer the cell suspension into the same 15-ml
centrifuge tube.
4. Centrifuge the 15-ml tube at 180g at 4 °C for 5 min.
5. Aspirate the medium gently without disturbing the pellet and resuspend the cells in 2 ml of MSCGM.
6. Mix 50 µl of the cell suspension and 50 µl of trypan blue stain and count the number of cells by using a cell counter.
7. Adjust the concentration to 50,000 cells per ml with MSCGM.
8. Seed 10 ml of the cell suspension onto a 100-mm dish (5.0 × 105 cells per 100-ml dish) and incubate the dish at 37 °C in a
humidified atmosphere of 5% CO2.
9. Maintenance. Feed human MSC cultures 3–4 d after plating by replacing the old medium with 10 ml of prewarmed MSCGM.
After 4–6 d, the cells become 80% confluent (Fig. 2d). Passage the cells by using the same procedure described above in steps 1–8.
10. Collection. Aspirate all of the spent medium from an 80% confluent 100-mm dish of human MSCs and add 5 ml of PBS(−). Remove
the PBS(−), add 1 ml of trypsin-EDTA and incubate the mixture at 37 °C in a CO2 incubator until the cells are less than 90% detached
(2–6 min).
11. Once the cells have detached, add 2 ml of MSCGM to the dish.
12. Transfer the cells to a 15-ml tube. Wash the cells by centrifuging at 180g for 5 min at 4 °C, removing the supernatant and
resuspending the pellet in MSCGM. The minimal volume of MSCGM will depend on the number of dishes (0.5–1 ml per 100-mm dish).
13. Mix 50 µl of the cell suspension and 50 µl of trypan blue stain and count the number of cells with a cell counter.
14. Adjust the concentration to 200,000 cells per ml with MSCGM.
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406 | VOL.9 NO.2 | 2014 | NATURE PROTOCOLS
(viii) Allow the mice to recover for 7–14 d before transplantation.
(ix) Transplantation under the cranial window (Supplementary Video 3). Anesthetize the immunodeficient mouse by an i.p.
injection of ketamine/xylazine.
(x) Remove the coverslip with a needle (Fig. 3f,g).
(xi) Rinse the cortex with normal saline.
(xii) Remove a liver bud from the culture plate and place it on the cortex with a sterile spatula or pipette (Fig. 3h).
(xiii) Lower a circular coverslip to cover it, taking care not to introduce air bubbles (Fig. 3i).
(xiv) Seal the edge of the optical window to the skull with a compound of cyanoacrylate glue and dental cement (Fig. 3j).
The compound will also cover the exposed skull.
(B) Transplantation under kidney capsule
(i) Set up a surgical field within an open front hood equipped with a gas outlet from an isoflurane vaporizer.
(ii) Anesthetize a recipient mouse with isoflurane, and then shave its flank with electric shears. Shave an area larger than
that where the transplants will be performed.
(iii) Position the animal with the shaved flank face up. Spray the flank with 70% (vol/vol) ethanol. Prepare the mouse with
a surgical scrub of iodine tincture. Drape the mouse, allowing access to the shaved flank.
(iv) By using your fingers, localize the kidney through the skin of the mouse under anesthesia.
(v) Once the kidney is localized, use the scissors to make a 2-cm incision through the dermis directly above it in a
direction perpendicular to the spinal cord. This incision will expose the peritoneal wall.
(vi) With the scissors, make a 1-cm incision through the peritoneal wall in the same direction as the cut through the
dermal layer. It is important that the opening created by this incision be smaller than the kidney being exposed.
CRITICAL STEP If the opening is slightly smaller than the kidney, the kidney will squeeze through the opening and
rest stably without any additional manipulation or contact (Fig. 3k). This positioning is optimal for the subsequent
transplantation steps. If the opening is too big, the kidney will fall back into the peritoneal cavity, making it difficult
to complete subsequent steps.
(vii) Squeeze through the kidney and wet the surface of the exposed kidney with normal saline. Repeat this step every few
minutes or as needed to prevent the kidney capsule from drying out.
(viii) By using sharp tweezers, make a 0.2-cm incision through the kidney capsule across the anterior surface of the kidney,
moving from the left lateral side toward the right lateral side (Fig. 3l).
(ix) Use the tweezers to create a pouch between the kidney capsule and kidney parenchyma. It is important to make this
pouch as long and narrow as possible. Short, wide pouches are not ideal, because they allow the liver bud to escape
from the capsule. Long, narrow pouches allow the liver bud to be deposited toward the posterior end of the kidney
with minimal loss from the capsular pouch.
(x) Retrieve the liver bud from the culture plate with a sterile spatula and put it on the capsule near the pouch (Fig. 3m).
(xi) Slowly transfer the liver bud into the subcapsular pouch by using tweezers (Fig. 3n). Ensure that the liver bud is
transferred into the pouch. Do not fill the pouch with air or excess liquid (Fig. 3o).
(xii) Gently push the kidney back into the peritoneal cavity.
(xiii) Close the incision by applying sutures to the peritoneal wall and the dermal layer.
(xiv) Return the mouse to its cage. Mice should be kept warm with a heating pad beneath the cage or under a heating lamp
with their eyes protected until they fully recover from anesthesia.
(C) Transplantation onto the distal mesentery
(i) Set up a surgical field within an open front hood equipped with the gas outlet from the isoflurane vaporizer.
(ii) Anesthetize a recipient mouse with isoflurane and shave the mouse outside of the area where the transplants will be performed.
(iii) Make a 1-cm incision on the medial part of the abdomen with surgical scissors under anesthesia.
(iv) Gently expose the small intestine and surrounding mesentery from the abdomen with cotton swabs (Fig. 3p).
(v) Fold back the mesentery.
(vi) Place the liver bud just above the mesentery, wrapping the abundant blood vessels (Fig. 3q,r).
(vii) Apply a small drop of fibrin glue (Bolheal) to the surrounding blood vessel (Fig. 3s). Gently push the surface of the
liver bud with forceps to ensure the fixation of the transplant (Fig. 3t).
(viii) Suture the abdomen closed.
(ix) Return the mouse to its cage. Mice should be kept warm with a heating pad beneath the cage or under a heating lamp
with their eyes protected until they fully recover from anesthesia.
(D) Transplantation onto the proximal mesentery (adjacent to the portal vein)
(i) Set up a surgical field within an open front hood equipped with the gas outlet from the isoflurane vaporizer.
(ii) Anesthetize a recipient mouse with isoflurane and shave its flank with electric shears. Shave the mouse outside of the
area where the transplants will be performed.
(iii) Make an incision in the recipient by using forceps and scissors.
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NATURE PROTOCOLS | VOL.9 NO.2 | 2014 | 407
(iv) Use cotton swabs to pull out the small intestine from the abdomen.
(v) Gently expose the portal vein and surrounding tissues with tweezers.
(vi) Carefully remove the capsule over the portal vein with fine tweezers or microscissors (Fig. 3u).
CRITICAL STEP This step is important for the efficient engraftment of the liver bud after transplantation (Fig. 3v).
(vii) Retrieve the liver bud from the culture plate with a sterile spatula and place it onto the surrounding area of the
portal vein (Fig. 3w,x).
(viii) Apply a small drop of fibrin glue (Bolheal) to the surface of the liver bud. Gently push the surface of the liver bud with
forceps to ensure the fixation of the transplant (Fig. 3y).
(ix) Gently push the intestine back into the peritoneal cavity.
(x) Close the mouse by applying sutures to the peritoneal wall and the dermal layer.
(xi) Return the mouse to its cage. Mice should be kept warm with a heating pad beneath the cage or under a heating lamp
with their eyes protected until they fully recover from anesthesia.
41| House the mice and proceed to Step 42 (intravital imaging) at appropriate time points for your experiment; intravital
imaging analysis can be initiated from the day of transplantation, and it can be continued up to at least 6 months. Initial
vascularization of iPSC-LB should be observed after ~48 h. The vessel connection between the human and mouse should be
well visualized by fluorescence imaging at days 7–10 after transplantation.
Intravital imaging
42| Anesthetize the mouse prepared in Step 40 by an i.p. injection of ketamine/xylazine.
43| Remove stains from the window with cotton swabs.
44| Immobilize the mouse’s head by Scotch-taping it to a cover glass.
45| Conduct imaging analyses by using a stereomicroscope or TCS SP5 confocal microscope.
46| After imaging, gently detach the Scotch tape and cover glass from the mouse’s head.
? TROUBLESHOOTING
Troubleshooting advice can be found in Table 1.
TABLE 1 | Troubleshooting table.
Step Problem Possible reason Solution
11 Poor attachment of human
iPSCs to MEF-coated plates
Variability between
cell lines
Add ROCK inhibitor Y-27632 to the medium during passaging and for
the next 24 h after passaging
12 Decrease in pluripotency
after passaging
Breakup of human iPSC
colonies is not optimal
If the clumps are too big, the middle of the colony will differentiate,
disturbing the downstream endoderm specification. This differentiation
can be avoided by vigorous pipetting of the clumps during passaging
22 Suboptimal differentiation Variability between
cell lines
DE differentiation is the most important part of the protocol. Indeed,
artificial hepatocytes are systematically obtained as long as this step of
the protocol works efficiently. Thus, the optimization of the first part
of the protocol is essential. DE differentiation can then be monitored
using qPCR, immunocytochemistry and flow cytometry analyses. At least
60% of the cells should express an endodermal marker to ensure an
efficient hepatic differentiation
24, 33 Excessive cell death and
poor differentiation
Incorrect composition of
culture medium
Prepare new medium and check the concentration of cytokines
38 Failure of 3D tissue
formation
Insufficient Matrigel
pre-solidification
It is essential to solidify the Matrigel. Leave the Matrigel at 37 °C in an
incubator for at least over 10 min
Failure of endothelial
sprouting in iPSC-LBs
Inadequate quality of
each cell type
Avoid over-confluency and -passaging of each cell type. Regarding
HUVECs, use the fresh HUVECs within three or four passages
© 2014 Nature America, Inc. All rights reserved.
PROTOCOL
408 | VOL.9 NO.2 | 2014 | NATURE PROTOCOLS
TIMING
Steps 1–4 (day 0): ~30 min will be required to coat the dish and plate the MEFs
Steps 5–12 (day 1): the day of passaging of human iPSCs requires ~1 h
Step 13 (days 2–5): only a medium change is required; it should take 10–20 min every day
Steps 14–23 (day 6): ~3 h will be required to coat the dish and passage and plate the cells to begin the human iPSC
differentiation
Step 24 (days 7–12; correspond to days 2–7 after plating of differentiating iPSCs): ~10 min to add cytokines to the medium,
aspirate old medium and add fresh medium
Step 25 (days 13–15; correspond to days 8–10 after plating of differentiating iPSCs): ~10 min to add cytokines to the
medium, aspirate old medium and add fresh medium
Steps 26–37 (day 16): 2–4 h will be required to prepare the medium and plates and collect and coculture each lineage cell
line for self-organization
Step 38 (day 17 onward): ~10 min every day of the protocol will be required to aspirate old medium and add fresh medium
Step 39 (days 17 and 18): ~20 min for setup of time-lapse imaging
Step 40A: each step of preparation (i–vii) and transplantation (ix–xiv) should be performed within the first 30 min
of anesthesia
Step 40B: ~1 h will be required for transplantation procedures
Step 40C: ~30 min will be required for transplantation procedures
Step 40D: ~1 h will be required for transplantation procedures
Step 41 (day 18 onward (from 0 d after transplant)): variable, depending on the experiment
Steps 42–46: 30 min plus time required for imaging
Box 1 (day 13): ~1 h will be required to passage and plate cells for maintenance and experimental purposes
Box 1 (days 14 and 15): ~10 min every second day of the protocol will be required to add fresh cytokines to the medium,
aspirate old medium and add fresh medium
Box 2 (day 10): ~1 h will be required to passage and plate cells for maintenance and experimental purposes
Box 2 (days 11–15): ~10 min every 3–4 d of the protocol will be required to add fresh cytokines to the medium, aspirate old
medium and add fresh medium
ANTICIPATED RESULTS
By following the protocol above, ~1 × 106 HNF4A-positive hepatic endodermal cells (immature endodermal cells destined to
track the hepatic cell fate) can be generated from human iPSCs plated on a 6-cm plate (Fig. 2a). qPCR analysis should
demonstrate that the cells generated at day 9 of this protocol express an increased level of hepatic endoderm marker genes
such as FOXA2 and HNFA compared with undifferentiated iPSCs, whereas pluripotency markers NANOG and POU class 5
homeobox 1 (OCT4; officially known as POU5F1) or definitive endoderm markers chemokine (C-X-C motif) receptor 4 (CXCR4)
and cerberus 1, DAN family BMP antagonist (CER1) are significantly downregulated (Fig. 2b)14. Stable differentiation should
also be validated by immunostaining for HNF4A antigen.
For liver bud generation, human iPSC-HEs are cocultured with freshly grown HUVECs and MSCs (Fig. 2c,d). In a couple of
hours, cells initiate invagination, as seen in organogenesis (Fig. 2e,f), and form 3D liver buds after 72 h (Fig. 2g).
Successfully formed liver buds are grossly visible and typically 4–7 mm in diameter on a Matrigel-coated 24-well dish.
Confocal microscopy observation of the inner portion of the tissues shows endothelial sprouting with perivascular localiza-
tion of MSCs and homogenously distributed human iPSC-HEs through the use of fluorescent protein-labeled cells (Fig. 2h).
Importantly, gene expression analyses show a significant hepatic commitment of human iPSC-HEs, as evidenced by the
expression of early hepatic marker genes such as AFP, albumin (ALB), retinol binding protein 4, plasma (RBP4) and
transthyretin (TTR). Microarray profiling shows that the expression profiles of human iPSC-LBs at 4 d in culture more closely
resembles those of mouse E10.5 and E11.5 liver buds as compared with advanced fetal or adult livers. The FACS-based
quantitative characterization of human iPSC-LBs was described in our previous report14,21.
After the transplantation of an in vitro–grown human iPSC-LB, functional blood perfusion is initiated within 24–48 h
in vivo, as confirmed by macroscopic observation (Fig. 4a,b). The successful vascularization of the transplanted liver
bud largely relies on the in vitro quality of the HUVECs; hence, special care must be taken to avoid overpassaging or
overconfluency. The highly branched vessel network that is initially established by 2 d after transplantation will be
pruned, and it will mature into a more refined vasculature that is presumably organ-specific by 7–14 d after transplantation
(Fig. 4c). Intravital confocal microscopy shows the patency of these human vasculatures via a fluorescence-conjugated
dextran infusion into the tail vein (Fig. 4dg). The direct connection between human and mouse vessels can be visualized
live by the infusion of an Alexa Fluor 647–conjugated anti-mouse CD31 antibody (Fig. 4h).
To evaluate liver function, ELISAs on the serum of transplant recipient mice can be routinely used to demonstrate the
secretion of human-specific ALB and α-1-antitrypsin (A1AT). Generally, the detection of these proteins is possible from
© 2014 Nature America, Inc. All rights reserved.
PROTOCOL
NATURE PROTOCOLS | VOL.9 NO.2 | 2014 | 409
7–14 d after transplantation when a single liver bud is transplanted, and the total levels of these proteins should continue
to increase along with the transplant course. Furthermore, after 30–60 d, the mice will begin to show human-specific drug
metabolism, which is one of the major functions of the liver. After exposure to a specific drug such as ketoprofen or
debrisoquine, the formation of human-specific metabolites can be demonstrated in urine and serum samples by mass
spectrometry analysis. Although further studies must be needed, it is notable that mesenteric transplantation proximal to
the liver seems to offer improved functionality compared with the other multiple ectopic transplant models (Fig. 5).
We speculate from this result that the perfusion of blood from the portal circulation has an important role in supporting
the engraftment, maturation and maintenance of human iPSC-LBs in vivo, which improves hepatic functions. Taken together,
our human iPSC-LB transplant approach (i.e., organ bud transplant) may pave the way for regenerative medicine and future
industrial use.
Note: Any Supplementary Information and Source Data files are available in the
online version of the paper.
ACKNOWLEDGMENTS We thank F. Kawamata, A. Takahashi, S. Funayama,
N. Hijikata and N. Sasaki for kindly providing technical support, and
Y.-W. Zheng, Y. Ueno and all of the members of our laboratory for help with
several comments. We express our great thanks to Y. Sato, SAIKOU, Inc. for
the illustrations for the protocols (http://www.medicalillustration.jp/). This
work was supported by Grants-in-Aid of the Ministry of Education, Culture,
Sports, Science, and Technology of Japan to T.T. (nos. 24106510, 24689052),
N.K. (no. 22390260) and H.T. (nos. 21249071, 25253079). This work was also
supported by grants to H.T. from the Research Center Network for Realization of
Regenerative Medicine and the Strategic Promotion of Innovative Research and
Development program (S-innovation, no. 62890004) of the JST; by a Specified
Research Grant of the Takeda Science Foundation and a grant from the Japan
IDDM network to H.T.; and by a grant of the Yokohama Foundation for Advanced
Medical Science to T.T. K. Eto and H. Nakauchi (Institute of Medical Science,
University of Tokyo) provided TkDA3 iPSCs.
AUTHOR CONTRIBUTIONS T.T. conceived, designed and conducted the experiments,
analyzed the data and wrote the paper. R.-R.Z., H.K., M.K., E.Y. and M.E.
performed experiments and wrote the paper. N.K., K.S. and H.T. supervised
the project.
COMPETING FINANCIAL INTERESTS The authors declare no competing financial
interests.
Reprints and permissions information is available online at http://www.nature.
com/reprints/index.html.
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