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Supplemental Information
Generation of Engraftable Hematopoietic Stem Cells from Induced Pluripotent Stem Cells by way
of Teratoma Formation
Nao Suzuki1, 3*, Satoshi Yamazaki1, 2*, Tomoyuki Yamaguchi1, 2, Motohito Okabe1, Hideki Masaki2,
Satoshi Takaki3, Makoto Otsu1, and Hiromitsu Nakauchi1, 2
*
These authors contributed equally to this work.
1
Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of
Medical Science, University of Tokyo, Tokyo 108-8639, Japan
2
Japan Science Technology Agency, Exploratory Research for Advanced Technology (ERATO)
Nakauchi Stem Cell and Organ Regeneration Project, Tokyo 108-8639, Japan
3
Department of Immune Regulation Research Institute, National Center for Global Health and Medicine,
Chiba 272-8516, Japan
Contains 5 Supplemental Figures and figure legends and one Supplemental Table.
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Legends for Supplemental Figures
Figure S1. Engraftable HSCs were induced from LG-iPSCs through teratoma formation
(a) Phase change of LG-iPSC-derived GFP+ CD45+ cells in PB of teratoma-bearing mice through
administration of cytokines and OP9 cells. (b) Analysis of BM cells of teratoma-bearing mice through
administration of cytokines and OP9 cells 12 weeks post iPSC injection. (c, d) BM transplantation assay.
BM cells of teratoma-bearing mice were transplanted into irradiated mice. (c) The chimerism of
LG-iPSC-derived GFP+ hematopoietic cells/CD45+ cells in spleen and bone marrow of recipient mice
(n=4) 12 weeks post primary transplantation. (d) The percentage of GFP+ cells/HPCs (Lin-) or HSCs
(CD34-KSL) in BM of recipient mice (n=4) 12 weeks post primary transplantation. Error bar represent
SD.
Fugure S2. Engraftable HSCs were induced from G-iPSCs through teratoma formation
(a) Phase change of G-iPSC-derived GFP+ CD45+ cells in PB of teratoma-bearing mice through
administration of cytokines and OP9 cells. (b) Analysis of BM cells of teratoma-bearing mice through
administration of cytokines and OP9 cells 12 weeks post iPSC injection. (c) The chimerism of
G-iPSC-derived GFP+ hematopoietic cells/CD45+ cells in spleen and BM of recipient mice (n=6) 12
weeks post primary transplantation. (d) The percentage of GFP+ cells/HPCs (Lin-) or HSCs (KSL;
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short-term, CD34-KSL; long-term) in spleen and BM of recipient mice (n=6) at 12 weeks post primary
transplantation. Error bar represent SD. (e) Blood count of recipient mice at 12 months after first
transplantation and age matched wild type B6 mice (n=4). WBC, white blood count; RBC, red blood
count; PLT, platelet; red lines represent the minimum value; WT, Wild type mice; RC, recipient mice.
Figure S3. Establishment of gene-corrected mγc-iPSCs from X-SCID mice
Expression of GFP and murine gamma chain (γc) in mγc-iPSCs. B6 iPSCs were used as a negative
control.
Figure S4.
Induction of engraftable HSCs from hiPSCs through teratoma formation
(a) Numbers of mice in which hiPSC-derived cells were detected in teratoma-bearing mice or recipient
mice transplanted with hiPSC-derived HSCs. (b-d) Immunostaining of human β-globin in
hiPSC-derived glycophorinA+ erythrocytes isolated from PB of recipient mice (b) and human
erythrocytes in PB (c), and mouse erythrocytes in PB (d). (e-g) Wright-Giemsa stains of cytospin
preparations of hiPSC-derived glycophorinA+ erythrocytes isolated from PB of recipient mice (e) and
human erythrocytes in PB (f), and mouse erythrocytes in PB (g). Scale bars, 10μm.
Figure S5.
iPSC-derived hematopoietic cells including HSCs and HSC niche components were
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coexist in teratomas
(a-c) Immunostaining of CD45, c-Kit and HSC niche-cell markers GFAP(a), osteocalcin (b), and
VE-cadherin (c) in G-iPSC-derived teratomas. Scale bar, 75 μm. (d, e) Immunostaining of hCD45,
hCD34, and HSC niche-like cells in hiPSC-derived teratoma sections. HSC niche-like cell markers were
osteocalcin (a), VE-cadherin (b). Scale bar, 30μm. Arrowheads indicate CD45+ CD34+ cells nearby HSC
niche-like cells.
Table S1. Chimerism of iPS-derived hematopoietic cells in recipient mice 12 weeks after bone
marrow transplantation
T cell chimerism is the percentage of GFP+ CD3+/CD45+ cells, B cell chimerim is the percentage of
GFP+ B220+/CD45+ cells, and myeloid chimerism is the percentage of GFP+ Gr-1+ Mac-1+ /CD45+ cells.
(Mean ± s.e.m.; LG-iPSCs group, n=4; G-iPSCs group, n=6.)
iPSCs
Peripheral blood
Spleen
Bone marrow
T cell
B cell
Myeloid
T cell
B cell
Myeloid
T cell
B cell
Myeloid
LG-iPSCs
20.9 ± 5.1
25.7 ± 5.7
26.5 ± 8.2
15.0 ± 0.1
24.9 ± 0.9
21.8 ± 0.5
1.3 ± 0.1
6.0 ± 0.4
48.0 ± 1.6
G-iPSCs
0.3 ± 0.2
2.4 ± 1.6
2.5 ± 1.1
0.3 ± 0.1
1.8 ± 0.1
0.3 ± 0.1
0.2 ± 0.1
0.7 ± 0.1
2.4 ± 1.2
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