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48
acinar
(exocrine cells)
liver
islet
(endocrine cells)
duodenum
pancreas
duodenum
centroacinar / interlobular / main
terminal duct
duct
pancreatic duct
centroacinar / terminal duct
islet
β cell : insulin
α cell : glucagon
duct
δ cell : somatostatin
islet
microvascular network
acinar
Figure 1. Structure of the pancreas and pancreatic islet
(A) Structure of the pancreas. Pancreatic juice, including digestive enzymes, is secreted from the
exocrine cells and is released into the duodenum through pancreatic ducts. The acinar structure is
consisted of exocrine cells and connects to centroacinar / terminal duct, and the islet structure is
composed of endocrine cells. (B) Hematoxylin and eosin (H&E) staining of pancreas sections
from adult mouse. (C) Structure of the islet. The pancreatic islet is composed of several hormoneproducing cells such as β cells, α cells, and δ cells and microvascular network.
49
~ E8.5
Dorsal
E8.5
E9.5 ~ 12.5
E13.5
E14.5~
bud
rotate
Ventral
Pancreatic
region
Pancreatic
buds
Multipotent
progenitors
“Tip-trunk“
formation
Pancreatic structure
formation
Tip (Ptf1a+)
Trunk (Nkx6.1+)
Endocrine progenitors
(Ngn3+ cells)
NGN3 expression :
Tip
Acinar
Duct
Trunk
Islet of Langerhans
Figure 2. Pancreatic development in the mouse
(A) Pancreatic development in embryonic day (E) 8.5 to E14.5. Dorsal and ventral pancreatic
buds arise E8.5. From E9.5 to E12.5, Pdx1+/Ptf1a+/Nkx6.1+ multipotent pancreatic progenitor
cells expand. Each pancreatic bud forms a branching structure and is compartmentalized into the
tip-trunk region in E13.5. At around E14.5, the ventral pancreatic bud rotates to dorsally, and
these buds fuse to form one pancreas. (B) “tip-trunk” region development. The tip region
differentiates the exocrine acinar. The trunk region mostly becomes the duct structure, and some
cells differentiate endocrine cells cluster islet of Langerhans.
50
Day
hiPSCs
DE
PFG
PP
EP
12
20
Stage 1
Stage 2
Stage 3
Stage 4
Activin A
CHIR99021
SANT-1
Dorsomorphin
SB431542
EC23
Indolactam V
FGF10
SANT-1
Dorsomorphin
SB431542
EC23
Indolactam V
Repsox
SANT-1
Dorsomorphin
Repsox
DAPT
IGF
Extendin4
EGF
Figure 3. Summary of the four stepwise differentiation protocol from human iPS cells
hiPSCs, human induced pluripotent stem cells; DE, definitive endoderm; PFG, posterior foregut;
PP, pancreatic progenitor; EP, endocrine progenitor; FGF10, fibroblast growth factor 10; IGF,
insulin-like growth factor ; EGF, epidermal growth factor.
51
PP-stage
Isotype control
(APC)
PDX1
(APC)
CXCR4 / c-Kit
positive cells (%)
80
60
40
20
DE
PDX1 positive cells (%)
CXCR4
(APC)
100
Isotype control
(APC)
Autofluorescence
(FITC)
c-Kit
(PE-Cy7)
Autofluorescence
(FITC)
Isotype control
(APC)
EP-stage
Autofluorescence
(FITC)
Isotype control
(PE-Cy7)
Autofluorescence
(FITC)
DE-stage
100
80
60
40
20
PP
NGN3
(APC)
NGN3 positive cells (%)
100
80
60
40
20
EP
Figure 4. Differentiation efficiency of each stage by flow cytometry analysis
(A-C) Flow cytometry analysis of the stage specific markers were performed. Ratio of CXCR4
and c-Kit double positive cells in definitive endoderm (DE)-stage (A), PDX1 positive cells in
pancreatic progenitor (PP)-stage (B), NGN3 positive cells in endocrine progenitor (EP)-stage (C).
Each gate defined by isotype negative control. Error bar represents the mean + SD of four
independent experiments.
52
hIveNry
-hiPSCs
PFG
PP
EP
(NGN3+)
Islet
(INS+)
mCherry (+)
Venus (+)
EP stage
5年以内に雑誌等で刊行予定のため、非公開。
mCherry/Venus
DE
Figure 5. Double knock-in INS-Venus/NGN3-mCherry (hIveNry) hiPSC-line
(A) Schematic illustration of differentiate into pancreatic islets from hiPSCs using hIveNry cellline. In hIveNry cells, Venus and mCherry cDNA were inserted under the control of INS and
NGN3 promoter at the ATG of the coding region, respectively (Yamashita-Sugahara et al., 2016).
(B) The live cell picture of EP-stage cells differentiated from hIveNry hiPSCs by fluorescence
microscopy. Scale bars, 100 μm. hiPSCs, human induced pluripotent stem cells; DE, definitive
endoderm; PFG, posterior foregut; PP, pancreatic progenitor; EP, endocrine progenitor; NGN3,
Neurogenin3; INS, Insulin.
53
mCherry
Brightfield
NGN3-mCherry
NGN3mCherryhigh
NGN3
-mCherryhigh
NGN3mCherrylow
NGN3
5年以内に雑誌等で刊行予定のため、非公開。
-mCherrylow
NGN3
-mCherry(-)
NGN3mCherry(-)
Autofluorescence (APC)
pre-sorted
Figure 6. Purification of hiPSC-derived EP cells by using NGN3-mCherry reporter
(A) Flow cytometry analysis of NGN3-mCherry expression was performed in EP-stage cells
derived from hiPSCs. EP-stage cells were divided into the following three fraction by expression
of mCherry, NGN3-mCherry(-), NGN3-mCherrylow, and NGN3-mCherryhigh. (B) The live cell
picture on day 1 in suspension culture after cell sort by fluorescence microscopy. Left panels,
mCherry fluorescence; right panels, brightfield. Scale bars, 100 μm.
54
Relative expression
( /GAPDH, pre-sorted=1)
NGN3
4.5
3.5
2.5
1.5
0.5
NEUROD1
＊＊
3.5
2.5
1.5
0.5
NKX2.2
3.5
2.5
1.5
0.5
＊＊
5年以内に雑誌等で刊行予定のため、非公開。
NGN3-mCherry
NGN3-mCherry
NGN3-mCherry
Relative expression
( /GAPDH, pre-sorted=1)
PDX1
2.5
1.5
0.5
NGN3-mCherry
Figure 7. Gene expression analysis of EP and PP markers
(A & B) mRNA expression was analyzed by qRT-PCR and shown are the relative expression
levels of the EP marker gene NGN3, NEUROD1, and NKX2.2 (A) and the PP gene PDX1 (B), in
pre-sorted, NGN3-mCherry(-), NGN3-mCherrylow, and NGN3-mCherryhigh cells. Expression level
of each gene is shown as relative expression level of averaged pre-sorted sample as 1.0. Error bar
represents the mean + SD of 4 independent experiments. Statistical analysis was performed using
the Turkey-Kramer test. * and **, significantly different from each of the other three fractions. *P
< 0.05, ** P < 0.01.
55
Day
EP
islet
10
Stage 5
DAPT
SANT1
Repsox
IGF
Ex-4
GLP1
5年以内に雑誌等で刊行予定のため、非公開。
NGN3
-mCherryhigh
Brightfield
INS-Venus
pre-sorted
NGN3
-mCherrylow
Figure 8. Differentiation of EP cells into pancreatic islet-like structures
(A) Summary of the differentiation protocol from EP cells to islets. (B) Representative images of
hiPSC-derived islets from pre-sorted, NGN3-mCherrylow, or NGN3-mCherryhigh cells with INSVenus. Scale bars = 100 μm. EP, endocrine progenitor.
56
INS
Relative expression
( /GAPDH, pre-sorted=1)
3.5
SST
GCG
4.5
3.5
2.5
1.5
2.5
5年以内に雑誌等で刊行予定のため、非公開。
1.5
0.5
NGN3-mCherry
0.5
NGN3-mCherry
NGN3-mCherry
Figure 9. Expression analysis of endocrine markers
mRNA expression was analyzed by qRT-PCR and shown are the relative expression levels of the
endocrine marker gene INS (insulin), GCG (glucagon), and SST (somatostatin), in pre-sorted,
NGN3-mCherrylow, and NGN3-mCherryhigh cells. Expression level of each gene is shown as
relative expression level of pre-sorted sample as 1.0. Error bar represents the mean + SD of 3
independent experiments. Statistical analysis was performed using the Turkey-Kramer test. *,
significantly different from each of the other two fractions. *P < 0.05.
57
NGN3-mCherrylow NGN3-mCherryhigh
C-pep/GCG/SST
pre-sorted
+ DAPI
5年以内に雑誌等で刊行予定のため、非公開。
Figure 10. Expression of endocrine markers in islets derived from hiPSCs
Immunofluorescent staining of spheroids from pre-sorted, NGN3-mCherrylow, or NGN3mCherryhigh cells was performed with antibodies recognizing insulin precursor C-peptide (C-pep,
green), glucagon (GCG, red), and somatostatin (SST, blue), together with nuclear staining (white)
using 4'-6- diamidino-2-phenylindole (DAPI). Scale bars = 50μm.
58
250
250
250
C-peptide (pM / 2×106 cells)
pre-sorted
200
200
NGN3-mCherrylow
200
NGN3-mCherryhigh
Lot #2
Lot #3
150
150
150
100
100
Lot #1
100
5年以内に雑誌等で刊行予定のため、非公開。
5050
50
00
Glucose
pre-sorted
NGN3
NGN3
low
-mCherry -mCherryhigh
pre-sorted
NGN3
NGN3
low
-mCherry
-mCherryhigh
Figure 11. Insulin secretion from hiPSC-derived islets
Glucose-stimulated insulin secretion (GSIS) of pancreatic islets derived from pre-sorted, NGN3-
mCherrylow cells, or NGN3-mCherryhigh cells. C-peptides secreted in supernatant of culture with
indicated glucose concentrations for 1 hour were determined. The y-axis shows total secreted Cpeptide normalized to cell numbers. Left graph represents average of the 3 independent
experiments. Error bar represents the mean + SD of 3 independent experiments. Right graph
represent the secretion of each lot.
59
DE-stage
PP-stage
EP-stage
Autofluorescence
(FITC)
hiPSCs
Ki67 positive cells / all cells (%)
Ki67
(APC)
90
80
70
60
50
40
30
20
10
hiPSCs DE
PP
EP
Figure 12. Expression of Ki67, a cell proliferation marker, by flow cytometry analysis in
each stage
(A) Flow cytometry analysis of Ki67 expression in stepwise differentiation from hiPSCs to EPstage cells. (B) The percentage of cells positive for Ki67 in stepwise differentiation from hiPSCs
to EP-stage cells. Error bar represents the mean + SD of 4 independent experiments. hiPSCs,
human induced pluripotent stem cells; DE, definitive endoderm; PFG, posterior foregut; PP,
pancreatic progenitor; EP, endocrine progenitor.
60
Anti-NGN3
Ki67 positive cells / all cells (%)
Anti-Ki67
16
**
14
12
10
NGN3
Figure 13. Expression of Ki67 in EP-stage cells
(A) Flow cytometry analysis of NGN3 and Ki67 in EP-stage cells. (B) A comparison of the
percentage of Ki67 positive cells in NGN3(-) and NGN3(+). Error bar represents the mean + SD
of five independent experiments. Statistical analysis was performed using the Mann-Whitney U
test. **P < 0.01.
61
Cell sort
(EP-stage cells)
Passage
NGN3
-mCherryhigh
Day
-2
Culture condition
2D
12
3D
Stage 4
Culture medium
Cell number (x105 cells)
1.5
**
0.5
12
Days
Figure 14. Cell growth arrest of NGN3-mCherryhigh cells
(A) Schematic image of culture methods of NGN3-mcherryhigh cells for 12 days. NGN3-
mCherryhigh cells were sorted from EP-stage cells, cultured for 2 days on 2 dimensional (2D)
culture in Stage 4 medium, and passaged every 4 days on 3 dimensional (3D) cultured in Stage 4
medium. (B) Growth curve of NGN3-mCherryhigh cells for 12 days. Error bar represents the mean
± SD of 4 independent experiments. Statistical analysis was performed using the Mann-Whitney
U test. * and **, significantly different from day 0. *P < 0.05, ** P < 0.01.
62
Lentivirus vector
5’LTR
GOI
EF1a
Transduction
(Lentivirus)
SIN
Gene
SV40LT
hTERT
cMYC
BMI1
Passage
NGN3
-mCherryhigh
Day
Culture condition
-2
2D
12
3D
Culture medium
Stage 4
Figure 15. Structure of lentivirus vector and culture methods for the expansion of NGN3mCherryhigh cells
63
＊＊
Figure 16. Growth curve of NGN3-mCherryhigh cells expressing growth-promoting genes by
lentivirus
Error bar represents the mean ± SD of three independent experiments (SV40LT and GFP
(negative control)) and a single experiment (c-MYC, hTERT, and BMI1). Statistical analysis was
performed using the Student’s t test. * and **, significantly different from SV40LT of day 0. *P <
0.05, ** P < 0.01.
64
NGN3-mCherryhigh
Figure 17. Induction and removal of SV40LT expression in NGN3-mCherryhigh cells by using
SV40LT-loxP and LV-Cre lentivirus vectors
65
Figure 18. Growth curve of NGN3-mCherryhigh cells expressing SV40LT genes by lentivirus
Blue line denotes cells transduced with SV40LT-loxP, and the black line denotes these without
transduction. Error bar represents the mean ± SD of five independent experiments.
66
Relative expression
(/GAPDH, passage 0 = 1)
NGN3
NKX2.2
1.2
1.2
1.4
1.2
0.8
0.8
0.6
0.6
0.8
**
0.4
0.4
0.2
0.2
Passage
Relative expression
(/GAPDH, passage 0 = 1)
NEUROD1
**
0.4
0.2
0 1 2 3 4 5
0 1 2 3 4 5
SUSD2
PDX1
1.4
1.2
2.5
0.6
0 1 2 3 4 5
0.8
0.6
0.4
1.5
0.5
0.2
Passage
0 1 2 3 4 5
0 1 2 3 4 5
Figure 19. Expression of EP markers in expanded NGN3-mCherryhigh cells
qRT-PCR analysis of endocrine progenitor markers (NGN3, NEUROD1, and NKX2.2) and an
endocrine progenitor surface marker SUSD2 and a pancreatic progenitor marker PDX1 in
expanded NGN3-mCherryhigh cells during five passages. Expression level of each gene is shown
relative expression level of averaged passage 0 samples as 1.0. Error bar represents the mean +
SD of 3 independent experiments. Statistical analysis was performed using the Student’s t test. *
and **, significantly different from passage 0. *P < 0.05, ** P < 0.01.
67
Day
+ SV40LT-loxP
± LV-Cre
NGN3-mCherryhigh
Expanded
NGN3-mCherryhigh
－28 ～ －14
Culture condition
2D
3D
10
3D
Stage 4
Culture medium
Figure 20. Schematic image of culture method of expanded NGN3-mcherryhigh cells treated
with or without LV-Cre.
After expansion of NGN3-mCherryhigh cells, cultured for 1 day on 2D culture in Stage 4 medium
to transduce LV-Cre, and cultured 9 days on 3D cultured in Stage 4 medium.
68
Non-transduced
Cell number
SV40LT-loxP (no Cre)
SV40LT-loxP + LV-Cre
SV40LT
Figure 21. Expression of SV40LT estimated by flow cytometric analysis
Flow cytometric analysis of SV40LT expression with or without LV-Cre induction shown by
histogram plotting. Black box indicates negative control experiments (Non-transduced of
lentivirus in NGN3-mCherryhigh cells), blue box and orange box indicate transduced without or
with LV-Cre, respectively.
69
Cell number (x105 cells)
3.5
Control
LV-Cre
2.5
1.5
0.5
10
Days
Figure 22. Growth curve of NGN3-mCherryhigh cells treated with or without LV-Cre
Orange line donates transduced with LV-Cre, and blue line denotes without lentivirus transduction
(Control). Error bar represents the mean ± SD of three independent experiments.
70
LV-Cre
Brightfield
NGN3
-mCherry
Control
Figure 23. Representative images of NGN3-mCherryhigh cells treated with or without LV-Cre
The live cell picture on day 10 in suspension culture after transduced with or without LV-Cre.
Upper, mCherry fluorescence; lower, brightfield. Scale bars, 100 μm.
71
Relative expression
(/GAPDH, control=1)
NGN3
10
**
NEUROD1
3.5
1.5
Control LV-Cre
2.5
NKX2.2
0.5
Control LV-Cre
Control LV-Cre
Figure 24. Transduction of LV-Cre rescues expression of endocrine-lineage commitment
genes in NGN3-mCherryhigh cells
qRT-PCR analysis of endocrine progenitor markers NGN3, NEUROD1, and NKX2.2 in NGN3mCherryhigh cells transduced with LV-Cre compared with the cells without Cre. Expression level
of each gene is shown relative expression levels of averaged control sample as 1.0. Error bar
represents the mean + SD of five independent experiments. Statistical analysis was performed
using the Mann-Whitney U test. *P < 0.05, **P < 0.01.
72
NGN3-mCherryhigh
+ SV40LT-loxP
Islets (INS+)
± LV-Cre
Day -2
-1
10
Culture condition
2D
3D
Culture medium
Stage 4
Stage 5
C-peptide (pM / 2×106 cells)
400
400
400
Lot #1
300
300
300
Lot #2
Lot #3
200
200
200
100
100
100
00
2.5mM
25mM
Glucose
2.5mM
25mM
Glucose
Figure 25. Pancreatic islet differentiation from NGN3-mCherryhigh cells immediately after
LV-Cre transduction
(A) Schematic differentiation into hiPSC-derived islets from expanded NGN3-mCherryhigh cells
immediately after LV-Cre transduction. (B) Glucose challenge test; islets derived from expanded
NGN3-mCherryhigh cells immediately after LV-Cre infection were cultured in the presence of
indicated glucose concentrations for 1 hour and the amounts of C-peptide in the supernatant were
examined. The y-axis shows total secreted C-peptide normalized by cell numbers. Left graph
represents average of the 3 independent experiments. Error bar represents the mean + SD of 3
independent experiments. Right graph represent the secretion of each lot.
73
NGN3-mCherryhigh
+ SV40LT-loxP
C-peptide (pM / 2×106 cells)
400
400
300
300
400
Control
LV-Cre
300
Lot #1
Lot #2
Lot #3
200
200
200
100
100
100
Glucose
Control
LV-Cre
Control
LV-Cre
Figure 26. Differentiation into pancreatic islets from expanded NGN3-mCherryhigh cells
(A) Schematic image of differentiation into hiPSC-derived islets from expanded NGN3mCherryhigh cells with or without LV-Cre. (B) Glucose challenge test; islets derived from
expanded NGN3-mCherryhigh cells with or without transduction with LV-Cre were cultured using
indicated glucose concentrations for 1 hour and the amounts of C-peptide in the supernatant were
examined. The y-axis shows total secreted C-peptide normalized by cell numbers. Left graph
represents average of the 3 independent experiments. Error bar represents the mean + SD of 3
independent experiments. Right graph represent the secretion of each lot.
74
EP cells
(NGN3+)
Islet differentiation
(Stage 5)
...
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