Stem Cell Reports

Article

Generation of Vascular Endothelial Cells and Hematopoietic Cells by

Blastocyst Complementation
Sanae Hamanaka,1 Ayumi Umino,1 Hideyuki Sato,1 Tomonari Hayama,1,3 Ayaka Yanagida,1,4
Naoaki Mizuno,1 Toshihiro Kobayashi,1,5 Mariko Kasai,1 Fabian Patrik Suchy,2 Satoshi Yamazaki,1
Hideki Masaki,1 Tomoyuki Yamaguchi,1,* and Hiromitsu Nakauchi1,2,*
1Division of Stem Cell Therapy, Distinguished Professor Unit, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku,

Tokyo 108-8639, Japan

2Institute for Stem Cell Biology and Regenerative Medicine, Department of Genetics, Stanford University School of Medicine, 265 Campus Drive, Stanford,

CA 94305, USA
3Present address: Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland,

OR 97239, USA
4Present address: Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Tennis Court Road,

Cambridge CB2 1QR, UK

5Present address: Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, 38 Nishigonaka Myodaiji, Okazaki, Aichi 444–8585,

Japan
*Correspondence: tomoyama@ims.u-tokyo.ac.jp (T.Y.), nakauchi@stanford.edu (H.N.)
https://doi.org/10.1016/j.stemcr.2018.08.015

SUMMARY

In the case of organ transplantation accompanied by vascular anastomosis, major histocompatibility complex mismatched vascular
endothelial cells become a target for graft rejection. Production of a rejection-free, transplantable organ, therefore, requires simultaneous
generation of vascular endothelial cells within the organ. To generate pluripotent stem cell (PSC)-derived vascular endothelial cells, we
performed blastocyst complementation with a vascular endothelial growth factor receptor-2 homozygous mutant blastocyst. This mutation
is embryonic lethal at embryonic (E) day 8.5–9.5 due to an early defect in endothelial and hematopoietic cells. The Flk-1 homozygous
knockout chimeric mice survived to adulthood for over 1 year without any abnormality, and all vascular endothelial cells and hemato-
poietic cells were derived from the injected PSCs. This approach could be used in conjunction with other gene knockouts which induce
organ deficiency to produce a rejection-free, transplantable organ in which all the organ’s cells and vasculature are PSC derived.

INTRODUCTION Nearly all pancreatic cells, including exocrine and endo-

crine cells, were derived from the injected PSCs. However,
Two key constraints to successful treatment using human cells originating from non-pancreatic lineages, such as
organ transplantation are graft rejection and organ blood vessels and stromal cells, were chimeric for both blas-
shortage. Autologous tissue stem cells or cell sheets gener- tocyst-derived cells and PSC-derived cells (Kobayashi et al.,
ated from patient-derived induced pluripotent stem cells 2010). We had similar results when targeting the kidney
(iPSCs) have been transplanted to eliminate rejection-asso- with blastocyst complementation—the renal lineage cells
ciated problems. Unfortunately, these cell therapies are were derived from injected PSCs, whereas non-renal line-
often not adequate to repair or replace a failing organ ages within the kidneys were chimeric (Usui et al., 2012).
(Assawachananont et al., 2014; Takebe et al., 2013). A major histocompatibility complex (MHC) mismatch of
Blastocyst complementation can be used to generate the vascular endothelial cells (a monolayer of cells lining
entire organs derived from pluripotent stem cells (PSCs). the lumen of vessels) will elicit hyperacute rejection
In this method, PSCs are injected into a blastocyst that against the blood vessel endothelium in the transplanted
has been genetically modified to prevent development of organ. Hyperacute rejection often occurs within 24 hr
a targeted organ (Kobayashi et al., 2010; Matsunari et al., and is initiated by recipient’s natural antibodies against
2013; Usui et al., 2012; Yamaguchi et al., 2017). The PSCs the antigens present in the graft’s vascular endothelial cells.
integrate into the growing embryo to form a chimeric ani- After recognition of the antigens, the complement and
mal; however, the targeted organ develops exclusively from coagulation systems are activated, resulting in inflamma-
the injected PSCs. tion and vascular occlusion. This will cause the graft to
We first successfully generated PSC-derived organs using rapidly necrose. Between 6 days and 3 months after trans-
blastocyst complementation in 2010. To target the plantation, acute rejection may occur, which is also caused
pancreas we knocked out Pdx1, resulting in apancreatic by an MHC mismatch of the vascular endothelial cells.
mice with severe hyperglycemia that died within a week Acute rejection caused by effector T cells, antibodies, and
after birth. We rescued this phenotype by injecting mouse activated T cells will directly lyse the graft’s vessels and pro-
or rat PSCs into the Pdx1 knockout (KO) mouse blastocysts. duce cytokines that recruit and activate inflammatory cells

988 Stem Cell Reports j Vol. 11 j 988–997 j October 9, 2018 j ª 2018 The Authors.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
(Platt et al., 1990, 1991). Therefore, in the context of blas- mPSCs Can Rescue Flk-1 KO Lethality by Blastocyst
tocyst complementation, it is necessary to generate organs Complementation
together with vascular endothelial cells in the blood vessels To generate blood vessels in Flk-11173F/1173F mice, blasto-
from a patient’s iPSCs to prevent organ rejection. cysts and morulae obtained from an intercross of Flk-
In this study, we aimed to generate blood vessels contain- 1+/1173F mice were injected with EGFP or KuO-labeled
ing entirely PSC-derived vascular endothelial cells by blas- miPSCs or mouse embryonic stem cells (mESCs). A total
tocyst complementation. In mice, vasculogenesis is of 105 chimeric mice were born and matured to adults
initiated from the yolk sac blood islands at E7.5 and is with no remarkable abnormalities. Of these, 11 were Flk-
dependent on several key factors. Disrupting vascular endo- 11173F/1173F, indicating that mPSCs can contribute to vascu-
thelial growth factor receptor 2 (VEGFR2/Flk-1/KDR) inhibits logenesis and rescue the Flk-1 KO phenotype (Table 1).
vasculogenesis due to impaired endothelial and hemato- Immunofluorescence was used to analyze the distribu-
poietic cell development, resulting in embryonic lethality tion of miPSC-derived cells in each tissue. In the Flk-
around E9.0 (Shalaby et al., 1995, 1997). Since Flk-1 mutant 11173F/1173F chimeric mice, PECAM1+ vascular endothelial
mice (Flk-11173F/1173F) represent the same phenotype as a cells in the aorta, kidney, lung, and heart were entirely
Flk-1 KO mice, Flk-1 mutant blastocysts were used as our derived from miPSCs. Other structures not of the vascular
host embryo for blastocyst complementation (Sakurai endothelial lineage, such as vascular smooth muscle in
et al., 2005). the aorta, Bowman’s capsule and renal tubule in the kidney,
bronchioles and alveoli in the lung, and myocardium in
the heart, were a composite of host and donor derivatives
RESULTS (Figures 2A, 2C, 2E, 2F, S2A, S2B, and S2E). In expected
contrast, all structural components, including vascular
Flk-11173F/1173F miPSC-Derived Cells Cannot endothelial cells of the aorta, kidney, lung, and heart in
Contribute to PECAM1-Expressing Vascular Flk-1+/1173F chimeric mice, were a composite of host and
Endothelial Cells in Adult Mice donor derivatives (Figures 2B, 2D, 2G, 2H, S2C, S2D,
Although the Flk-1 homozygous mutant (or Flk-11173F/1173F) and S2F).
mouse is well studied at early embryonic stages, because of To further analyze the contribution of mPSCs to vascular
embryonic lethality at E8.5–9.5, the role of Flk-1 in vasculo- endothelial cells, we performed fluorescence-activated cell
genesis from E9.5 to adulthood is unclear. To address this sorting (FACS) analysis of enzymatically dissociated tissues
issue, we generated chimeric mice by injecting enhanced from adult chimeric mice.
green fluorescent protein (EGFP)-marked Flk-11173F/1173F In Flk-11173F/1173F chimeric mice, almost 100% of the
mouse-induced PSCs (miPSCs) into wild-type (WT) mouse vascular endothelial cell population (CD45/PECAM1+)
blastocysts (Figures S1A and S1B). We first analyzed the of the lungs, kidneys, and aortas were EGFP positive (Fig-
contribution of Flk-11173F/1173F cells to blood vessels in ures 3A, S3A, and S3B), whereas populations other than
E13.5 embryos (Figures 1A and 1B). The immunofluores- vascular endothelial cells (CD45/PECAM1 population),
cent staining of a section of intestine with relatively the cells in the vessel wall, or the perivascular cells
high chimerism revealed that the EGFP-expressing (CD45/PECAM1/platelet-derived growth factor receptor
Flk-11173F/1173F iPSC-derived cells did not express platelet beta [PDGFR b+]) and the overall cells in the lungs, kidneys,
endothelial cell adhesion molecule 1 (PECAM1) (arrow) and aortas were chimeric (39.8%–95.7%) (Figures 3B, 3C,
(Figure 1A). In addition, flow cytometric analysis of fetal S3A, and S3B).
liver showed that the CD45 and PECAM1+ (also known These results indicate that the mPSCs could complement
as CD31) vascular endothelial cells did not express the vasculogenic niche and create functional blood vessels
EGFP (Figure 1B). Next, in order to analyze the contribution in Flk-11173F/1173F mice. Moreover, the vascular endothelial
of Flk-11173F/1173F iPSCs in adult chimeric mice, we per- cells in blood vessels were completely derived from mPSCs,
formed immunofluorescent analysis of a pancreas that whereas the other components were composed of host and
showed relatively high chimerism and found that EGFP+ donor-derived cells. This means whole chimera mouse
Flk-11173F/1173F iPSC-derived cells did not express PECAM1 body was not composed of almost 100% donor cells and
(Figures 1C, S1C, and S1D). successfully complemented specific tissue by blastocyst
These results indicate that Flk-11173F/1173F iPSC-derived complementation. Although we tried to generate rat
cells cannot contribute to vasculogenesis or angiogenesis vascular endothelial cells in mice by injecting rat PSCs
from the early embryo to adulthood. Thus, the Flk- into Flk-11173F/1173F mouse blastocysts, we could not obtain
11173F/1173F mouse is a suitable host animal for blastocyst live Flk-11173F/1173F fetuses at E13.5 and beyond. However,
complementation when generating PSC-derived blood at E9.5, live Flk-11173F/1173F interspecies chimeric fetuses
vessels. were found at Mendelian ratios (Table S1).

Stem Cell Reports j Vol. 11 j 988–997 j October 9, 2018 989

 A
GFP/ PECAM1/ DAPI GFP, Flk-11173F/1173F PECAM1 DAPI

B CD45(-) gate

PECAM1
CD45

3.7 9.6 0.9 0.00

85.6 1.1

0.5
PECAM1 EGFP

C GFP/ PECAM1/ DAPI GFP, Flk-11173F/1173F PECAM1 DAPI

Figure 1. Phenotype of Vasculogenesis in Flk-11173F/1173F iPSC-Derived Chimeric Mice

(A) Immunohistological analysis of vascular endothelial cells in embryo of Flk-11173F/1173F iPSC-derived chimeric mouse at E13.5. Sections
were stained with antibodies against GFP for Flk-11173F/1173F iPSC-derived cells, and PECAM1 for endothelial cells, and cell nuclei were
stained with DAPI. The vascular endothelia are indicated (arrows).
(B) Flow cytometry analysis of vascular endothelial cells in fetal liver. Fetal liver cells were stained with antibodies against CD45 and
PECAM1. Representative results from n = 8 independent experiments are shown.
(C) Immunohistological analysis of sections obtained from pancreas. Sections were stained with antibodies against GFP for Flk-11173F/1173F
iPSC-derived cells, antibodies against PECAM1 for endothelial cells (arrows) and DAPI for nuclear counterstaining. Lower panels show
higher magnification.
Scale bars: 50 mm (A) and 100 mm (C).

990 Stem Cell Reports j Vol. 11 j 988–997 j October 9, 2018

Table 1. Generation of Flk-11173F/1173F Chimera Mice by DISCUSSION
Blastocyst Complementation
Here we successfully used blastocyst complementation to
No. (%) of
Offsprings Flk-1 Genotype (%) generate mPSC-derived vascular endothelial cells and he-
Injected 1173F/ matopoietic cells, including HSCs.
Cell Lines Alive Chimeras +/+ +/1173F 1173F NDa In order to generate Flk-11173F/1173F chimeric mice, fluo-
GT3.2 48 37 (77) 13 (35) 20 (54) 4 (11) 0 (0) rescently labeled WT mPSCs were injected into blasto-
cysts/morulae obtained by intercross of a Flk-1+/1173F male
b
KuOmiPS 20 13 (65) 9 (69) 3 (23) 1 (8) 0 (0) with a Flk-1+/1173F female mouse. The birth rate of Flk-
SGE2 56 55 (98) 14 (25) 18 (33) 6 (11) 17 (31) 11173F/1173F chimeric mice was distorted from Mendelian
expectations. This distortion was also observed in E13.5
Total 124 105 (85) 36 (34) 41 (39) 11 (11) 17 (16)
to E15.5 chimeric fetuses (4/90 = 4.4%). We previously
a
Not determined. found that the birth rate of Pdx1/ chimeric mice obey
b
Neonate. Mendel’s law (Kobayashi et al., 2010), suggesting that
complementation efficiency may depend on the targeted
gene. For example, although the size of the pancreas may
All Hematopoietic Stem Cells in the Flk-11173F/1173F be restricted by the number of cells present in the pancre-
Chimeric Mouse Were Generated from Donor mPSCs atic anlage, we previously observed that a small pancreas
It was previously reported that the Flk-11173F/1173F mouse is enough to maintain blood glucose level (data not
has an embryonic lethal phenotype due to not only shown), indicating that the contribution rate of PSCs to
impaired vasculogenesis, but also deficient primitive pancreatic anlage might not affect the birth rate of comple-
hematopoiesis at E8.5 to E9.5. Therefore, we analyzed mented mice. On the other hand, Flk-1 is important for the
the contribution of mPSCs in CD45+ hematopoietic development of multiple tissues that are dispersed
cells to confirm whether the hematopoietic cells are throughout the whole body, including vascular endothelial
also completely derived from injected mPSCs in Flk- cells, hematopoietic cells, and smooth muscle cells. Hence,
11173F/1173F chimeric mice. FACS analysis of peripheral the contribution rate of PSCs to the vascular endothelial
blood mononuclear cells (PBMCs) revealed that all line- cell anlage might affect the survival of the developing
ages of hematopoietic cells were almost 100% EGFP mouse.
positive in Flk-11173F/1173F chimeric mice, whereas he- Although we successfully generated entirely PSC-derived
matopoietic cells in Flk-1+/1173F chimeric mice were vascular endothelial cells, the vessel wall and the perivascular
chimeric. Similar results were obtained by the FACS anal- cells, which include smooth muscle cells, pericytes, and renal
ysis of splenocytes (Figures 4A, S4A, and S4B). We also mesangial cells, were chimeric because those tissues can
analyzed the percentage of EGFP+ cells in, c-Kit+, Sca-1+, develop from both host neural crest and Flk-1+ mesoderm
and lineage (KSL) hematopoietic progenitor and stem cells (Hirschi and Majesky, 2004; Nakamura et al., 2006).
cells. We found that the chimerism of EGFP+ cells in the When performing blastocyst complementation with Flk-
KSL population was 100% in Flk-11173F/1173F chimeric 11173F/1173F mouse embryos, all hematopoietic cells were
mice and chimeric in Flk-1+/1173F chimeric mice (Figures entirely derived from the injected mPSCs. Primitive hema-
4B, S4C, and S4D). Furthermore, to analyze the reconsti- topoiesis is initiated in the yolk sac blood island at E7.5.
tution ability of miPSC-derived hematopoietic stem cells The site of hematopoiesis is then migrated to the aorta-
(HSCs) generated in Flk-11173F/1173F chimeric mice, we per- gonad-mesonephros (AGM) region, after which definitive
formed transplantation of the Flk-11173F/1173F chimera’s hematopoiesis is initiated around E10.5. Previous studies
whole bone marrow to sub-lethally irradiated recipient showed that HSCs first emerge from vascular endothelial
mice (n = 4) and analyzed the engraftment rate. The trans- cells in the dorsal aorta at E10.5 (Bertrand et al., 2010; Bois-
planted cells differentiated into three hematopoietic line- set et al., 2010; Herbert et al., 2009; Kissa and Herbomel,
ages, including B cell, T cell, and myeloid cells. The 2010). When performing blastocyst complementation
percentage of EGFP+ cells in hematopoietic cells was with Flk-11173F/1173F mouse embryos, the HSCs likely
98%; in B cells was 100%; in T cells was 92.3%; and in emerged from the complemented vascular endothelial
myeloid cells was 98.9% (Figures 4C and 4D). niche, resulting in generation of both PSC-derived vascular
These results indicate that mPSCs can complement endothelial cells and hematopoietic cells. Because residual
both the hematopoietic and vasculogenic niche, gener- hematopoietic cells left in a graft might induce graft versus
ating entirely mPSC-derived HSCs and hematopoietic host disease (GVHD) after transplantation, simultaneous
cells in Flk-11173F/1173F chimeric mice by blastocyst generation of tissue and hematopoietic cells from PSCs
complementation. might further reduce the risk of GVHD.

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 (legend on next page)

992 Stem Cell Reports j Vol. 11 j 988–997 j October 9, 2018

Figure 3. Flow Cytometry Analysis of the EGFP-Expressing, miPSC-Derived Cells in the Vascular Endothelium and Pericytes of Each
Organ
Ratio of donor cell contribution in lung, kidney and aorta of Flk-11173F/1173F or Flk-1+/1173F chimeric mice were analyzed using antibodies
against CD45, PECAM1, and PDGFRb.
(A) Vascular endothelial cells (CD45/PECAM1+) were gated and analyzed for EGFP expression.
(B) Non-vascular endothelial cells (CD45/PECAM1) were gated and analyzed for EGFP expression.
(C) Pericytes (CD45/PECAM1/PDGFRb+) were gated and analyzed for EGFP expression.
Red line, Flk-11173F/1173F chimeric mouse; blue line, Flk-1+/1173F chimeric mouse; gray dashed-line, C57BL/6 mouse shown as a control.
Three biological replicates were performed.

Although most of the PBMCs from Flk-11173F/1173F vascular system complemented Flk-11173F/1173F chimera
chimeric mice were EGFP positive, FACS analysis revealed mouse were 100% EGFP positive. Moreover, EGFP-negative
that 0.2% to 1.3% of hematopoietic cells were EGFP nega- hematopoietic cells were also observed in peripheral blood
tive. We found that the HSCs in the bone marrow of of the EGFP transgenic mouse, which is the origin of donor

Figure 2. Immunohistological Study of Chimeric Mice at Adult Stage

(A–D) The distribution of iPSC-derived cells in sections of aorta and kidney (E–H). Sections were stained with antibodies against
GFP, PECAM1, a-SMA, and DAPI. L, lumen; BV, blood vessel; G, glomerulus; T, tubules. Scale bars: 50 mm (C, D, F, and H) and 100 mm
(A, B, E, and G).

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 (legend on next page)

994 Stem Cell Reports j Vol. 11 j 988–997 j October 9, 2018

PSCs. These results indicate that gene silencing had et al., 1990, 1991). Chimeric animal models generated
occurred in EGFP-negative hematopoietic cells in periph- from Flk-1 deficient blastocysts thus provide a key step
eral blood of rescued Flk-11173F/1173F chimera mice. In toward in vivo generation of rejection-free organs for use
addition, there is another possibility that EGFP-negative in regenerative medicine.
hematopoietic cells were derived from the origin other
than HSCs, as previously described (Ginhoux et al., 2010;
Hoeffel et al., 2012). EXPERIMENTAL PROCEDURES
Because our goal is to generate tissues or organs in a xeno-
Animals
genic animal, we also tried to make rat vascular endothelial
C57BL/6NCrSlc, BDF1, ICR mice were purchased from SLC Japan
cells and hematopoietic cells in mice by injecting rat iPSCs (Shizuoka, Japan). Flk-1+/1173F mice were kindly provided by
into mouse blastocysts obtained by an intercross of Flk- Dr. M. Shibuya (University of Tokyo, Tokyo Medical and Dental
1+/1173F mice. Because Flk-11173F/1173F mice were embryonic University, and Jobu University) (Sakurai et al., 2005), and were
lethal at E8.5E9.5, even the incomplete complemented crossed with C57BL/6 or BDF1 mice. All experiments were per-
embryos were viable until E9.5 and follow Mendelian formed in accordance with the animal care and use committee
laws. Because incomplete complementation leads to imma- guidelines of the Institute of Medical Science, University of Tokyo.
ture vascularization and embryonic death after E9.5, the
birth rate of Flk-11173F/1173F chimeric mice and survival Culture of iPSCs/ESCs
rate of E13.5–E15.5 chimeric fetuses were distorted from Undifferentiated mouse iPSCs (GT3.2(XY) (Kobayashi et al., 2010),
Mendelian expectations in both intraspecies complemen- mESCs (SGE2) derived from EGFP transgenic mice, Flk-11173F/1173F
tation and interspecies complementation setting. The miPSCs, and Kusabira Orange (KuO) miPS) were maintained on
incomplete contribution of donor cells to the AGM region mitomycin C-treated mouse embryonic fibroblast (MEF) feeder
can induce embryonic death because the ischemic condi- cells in Dulbecco’s modified Eagle’s medium (Sigma, St. Louis,
MO) supplemented with 15% fetal bovine serum (Nichirei Biosci-
tion is caused by hematopoietic cell and vascular deficiency.
ence, Tokyo, Japan), 0.1 mM 2-mercaptoethanol (Invitrogen, San
Although mPSCs can rescue the Flk-11173F/1173F pheno-
Diego, CA, USA), 1% nonessential amino acids (Invitrogen),
type in mouse blastocysts, efficiency is low. Efficiency
1 mM sodium pyruvate (Invitrogen), 1% L-glutamine penicillin
could be immediately increased by abandoning the hetero- streptomycin (Sigma), and 1,000 U/mL of mouse leukemia inhibi-
zygous intercross approach, which results in only 25% Flk- tory factor (LIF; Millipore, Bedford, MA, USA).
11173F/1173F blastocysts. Instead, adopting nuclear transfer Flk-11173F/1173F miPSCs were generated from embryonic fibro-
technology or utilizing Flk-1-driven suicide genes would blasts harvested from an E13.5 Flk-11173F/1173F chimeric mouse
result in 100% Flk-1-deficient embryos. These approaches fetus (injected with GT3.2 iPSCs at the blastocyst stage). The iPSCs
are also useful for performing multiple gene disruptions. were generated by introducing three reprograming factors (Oct-
Currently, organs transplanted into human patients are 3/4, Sox-2, and Klf-4) and EGFP in all-in-one inducible lentiviral
supplied by human donors. These allogeneic grafts may vector (Ai-LV) (Yamaguchi et al., 2012).
be rejected and require each recipient to endure lifelong KuO miPS were generated from MEFs of KuO mice by intro-
ducing three reprograming factors (Oct-3/4, Sox-2, and Klf-4) in
immunosuppression. By simultaneously disrupting Flk-1
Ai-LV and CAG huKO IRES Puro (Hamanaka et al., 2013).
and genes required for organogenesis (e.g, Pdx1 for
riPST1-3 were generated from rat embryonic fibroblasts of Wistar
pancreas), autologous organs could be generated from rat by introducing three reprograming factors (Oct-3/4, Sox-2, and
patient-specific iPSCs via interspecies blastocyst comple- Klf-4) in Ai-LV (Hamanaka et al., 2011). Fri6.1 were generated from
mentation. These iPSC-derived organs would not be rat embryonic fibroblasts of F344 rat by introducing three reprog-
contaminated with xenogenic cells in the vascular endo- raming factors (Oct-3/4, Sox-2, and Klf-4) in pMx mOKS IRES
thelium, eliminating rejection-associated problems (Platt tdTomato and CAG huKO IRES Puro.

Figure 4. Flow Cytometry Analysis of Chimerism in miPSC-Derived Hematopoietic Cells

(A) Representative flow cytometric plots of EGFP and hematopoietic lineage marker expression in peripheral blood and splenocytes from
Flk-1 mutant chimeras. Three biological replicates were performed.
(B) Representative flow cytometric plots and gating for the c-Kit+Sca1+Lineage (KSL) fraction within the BM of Flk-1 mutant chimeras.
Three biological replicates were performed.
(C) Representative percentage of peripheral blood chimerism of 12 weeks after Flk-1 mutant chimera’s BM transplantation into lethally
irradiated four recipient mice.
(D) Time course of engraftment of complemented hematopoietic cells are shown. EGFP expression was analyzed in the peripheral blood 4,
8, and 12 weeks after transplantation.
Results are means ± SD of n = 4 (Flk-11173F/1173F; 4 weeks), n = 3 (Flk-11173F/1173F; 8 weeks), n = 2 (Flk-11173F/1173F; 12 weeks), n = 3
(Flk-1+/1173F; 4 weeks), n = 3 (Flk-1+/1173F; 8 weeks), and n = 3 (Flk-1+/1173F; 12 weeks) independent experiments.

Stem Cell Reports j Vol. 11 j 988–997 j October 9, 2018 995

 Maintenance of rat iPSCs has been previously described (Hama- (#100307), -CD45.2 (#109807) (BioLegend, San Diego, CA) and
naka et al., 2011). Briefly rat iPSCs were maintained on mitomycin -CD31 (PECAM1, #12-0311-81, eBioscience, San Diego, CA), allo-
C-treated MEF feeder cells in N2B27 medium (Invitrogen) supple- phycocyanin (APC)-conjugated anti-CD45 (#559864, BD Phar-
mented with 1,000 U/mL mouse LIF (Millipore, Bedford, MA, USA) mingen, Franklin Lakes, NJ), -CD45.1 (#17-0453-82, eBioscience)
or rat LIF (Rat ESGRO; Millipore ESG 2206), 1 mM GSK3 inhibitor and -CD140b (PDGFRb, #136008, BioLegend), phycoerythrin
CHIR99021 (Axon Medchem),1 mM MEK inhibitor PD0325901 cyanine7 (PECy7)-conjugated anti-CD45 (#103113, BioLegend,
(Stemgent or Wako), 1% L-glutamine penicillin streptomycin or #25–0451081, eBioscience), -Gr-1 (#108415) and -Mac-1
(Sigma). All PSC lines were authenticated by chimera formation. (CD11b, #101215), Pacific blue (PB)-conjugated anti-CD45
(#103126) and B220 (#163230) (BioLegend), brilliant violet (BV)-
Generation of Chimeric Mice and Genotyping of 421-conjugated anti-CD31 (#563356, BD Bioscience, San Jose,
iPSCs/ESCs Derived from Chimeric Mice CA), allophycocyanin cyanine7 (APC-Cy7)-conjugated anti-CD3
Chimeric mice were generated by injection of miPSCs into 3.5 days (#100221, BioLegend), and purified-CD16/32 (#553142, BD Phar-
post coitum (dpc) blastocysts and mESCs into 2.5 dpc morulae of mingen) antibodies, and propidium iodide for removing dead
WT or Flk-1+/1173F intercrossing embryos, followed by transfer cells. HSCs were stained with APC-conjugated anti-c-Kit (#17-
into host uteri as previously described (Kobayashi et al., 2010) 1171-81, eBioscience), PE-conjugated anti-Sca-1 (#108108), PB-
For genotyping, chimeric mouse fibroblasts were isolated from conjugated anti-Sca-1 (#108119) (BioLegend), and the lineage
E13.5–E14.5 mice and adult mice. GFP mouse fibroblasts were antibody-mix consisting of biotinylated anti-Gr-1 (#13593185),
sorted by cytometry using MoFlo or Aria cytometer (Beckman -Mac-1 (#13011285), -CD4 (#13004285), -Ter119 (#13592185),
Coulter, Fullerton, CA, USA). Genotypes of mice were confirmed -interleukin-7 receptor (#13127185) (eBioscience), and -CD8
using extracted genomic DNA from sorted GFP-expressing cells. (#13008185) and -B220 (#13045285) (BioLegend) antibodies.
PCR primers for amplification of Flk-1 were forward; 50 -GCCT The biotinylated antibodies were developed with streptavidin-
CTTCCAAGACAGTCT-30 and reverse; 50 -GAACCTTCAGCAGGT APC-Cy7 (#405208, BioLegend).
TTCCTATTTG-30 . After BspEI digestion of the PCR products After being washed with PBS containing 2% fetal calf serum
(729 bp), the products from the WT allele were not digested (FCS), stained cells were analyzed by flow cytometry using Cant
(729 bp), but the products from the Flk-11173F mutant allele were II or Aria II equipment (Becton-Dickinson, Franklin Lakes, NJ).
digested into 523 and 206 bp bands. For bone marrow (BM) transplantation, total BM cells were sus-
pended in 2% FCS in PBS; 2 3 107 total BM cells in 200 mL of me-
dium was injected into each of a group of lethally irradiated
Histological Analysis
C57BL/6 mice. Peripheral blood cells of the recipient mice were
For frozen sections, mouse tissues were fixed with 4% paraformal-
analyzed 4, 8, and 12 weeks after transplantation.
dehyde, washed with PBS, soaked in 30% sucrose and mounted in
optimal cutting temperature compound (Sakura Finetek, Tokyo,
Japan). Frozen sections were stained immunohistochemically. Statistics and Reproducibility
Each section was incubated with the primary antibody overnight The experiments were not randomized and the investigators were
at 4 C and with the secondary antibody for 1 hr at room not blinded to allocation during experiments and outcome assess-
temperature. ment. Statistical significance was calculated by F test and Student’s
The antibodies used for the primary antibody were antibodies t test (compare two groups) and the similarity to the Mendelian ra-
against EGFP (A11122, Invitrogen; and ab6673, Abcam, Cambridge, tio was analyzed by chi-squared test (with Excel and GraphPad
UK), platelet endothelial cell adhesion molecule-1 (PECAM1, Prism software). p < 0.05 was considered to be statistically signifi-
#550274, BD PharMingen, Franklin Lakes, NJ, USA), and a-smooth cant. Data are presented as mean ± SD.
muscle actin (aSMA, ab5694, Abcam). Secondary antibodies used
were Alexa 488-conjugated-goat anti-rabbit IgG (A11034), -donkey SUPPLEMENTAL INFORMATION
anti-rabbit IgG (A21206), -donkey anti-goat IgG (A11055), Alexa
Supplemental Information includes four figures and one table and
546-conjugated-goat anti-rat IgG (A11081), -donkey anti-rabbit
can be found with this article online at https://doi.org/10.1016/j.
IgG (A10040), Alexa 647-conjugated-goat anti-rabbit IgG
stemcr.2018.08.015.
(A21245), -chicken anti-rat IgG (A21472) (Invitrogen). After anti-
body treatment, sections were counterstained with 40 ,6-diami- AUTHOR CONTRIBUTIONS
dino-2-phenylindole (DAPI, Sigma) and mounted with fluorescence
mounting medium (Dako, Glostrup, Denmark). The sections S.H. and T.Y. designed this study, and wrote the manuscript. S.H.
were observed under fluorescence microscopy (BZ9000, Keyence, performed the experiments. A.U., M.K., and H.S. performed em-
Itasca, IL) bryo manipulation. A.U. and H.M. performed data analysis.
N.M., A.Y., T.H., and S.Y. performed animal experiments. T.K. per-
Flow Cytometry Analysis of iPSC/ESC-Derived formed establishment of miPSCs. F.P.S wrote the manuscript. H.N.
designed and supervised this study, and wrote the manuscript.
Chimeras and Bone Marrow Transplantation
Cells were dissociated with accutase (Innovative Cell Technolo-
ACKNOWLEDGMENTS
gies, San Diego, CA) for fetal liver, with accutase or trypsin for em-
bryonic fibroblast, and with collagenase IA for adult tissues. Cells We thank H. Tsukui and J. Ooehara for technical support, K. Okada
were stained with phycoerythrin (PE)-conjugated anti-CD33 for secretarial support, Dr. M. Shibuya for kindly providing

996 Stem Cell Reports j Vol. 11 j 988–997 j October 9, 2018

Flk-1+/1173F mice and Dr. M. Watanabe for critical advice in prepar- monocytes with a minor contribution of yolk sac-derived macro-
ing the manuscript. This work was supported by grants from Japan phages. J. Exp. Med. 209, 1167–1181.
Science and Technology Agency, Exploratory Research for Kissa, K., and Herbomel, P. (2010). Blood stem cells emerge from
Advanced Technology, Leading Advanced Projects for Medical aortic endothelium by a novel type of cell transition. Nature 464,
Innovation, AMED under grant number JP18gm0010002, 112–115.
KAKENHI grant number 17K10536, research grant for type 1
Kobayashi, T., Yamaguchi, T., Hamanaka, S., Kato-Itoh, M., Yama-
diabetes, Japan IDDM network and California Institute for Regen-
zaki, Y., Ibata, M., Sato, H., Lee, Y.S., Usui, J., Knisely, A.S., et al.
erative Medicine Grant Number LA1-06917. H.N. is a co-founder
(2010). Generation of rat pancreas in mouse by interspecific blasto-
and shareholder of iCELL Inc., ChimaERA Corporation, and
cyst injection of pluripotent stem cells. Cell 142, 787–799.
ReproCELL Inc.
Matsunari, H., Nagashima, H., Watanabe, M., Umeyama, K., Na-
Received: November 28, 2017 kano, K., Nagaya, M., Kobayashi, T., Yamaguchi, T., Sumazaki, R.,
Revised: August 22, 2018 Herzenberg, L.A., et al. (2013). Blastocyst complementation gener-
Accepted: August 22, 2018 ates exogenic pancreas in vivo in apancreatic cloned pigs. Proc.
Published: September 20, 2018 Natl. Acad. Sci. USA 110, 4557–4562.
Nakamura, T., Colbert, M.C., and Robbins, J. (2006). Neural crest
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