U.S. patent application number 16/589293 was filed with the patent office on 2020-04-02 for method for generating pancreatic bud cells and therapeutic agent for pancreatic disease containing pancreatic bud cells.
The applicant listed for this patent is Kyoto University. Invention is credited to Kenji OSAFUNE, Taro TOYODA.
Application Number | 20200102542 16/589293 |
Document ID | / |
Family ID | 1000004500216 |
Filed Date | 2020-04-02 |
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United States Patent
Application |
20200102542 |
Kind Code |
A1 |
OSAFUNE; Kenji ; et
al. |
April 2, 2020 |
METHOD FOR GENERATING PANCREATIC BUD CELLS AND THERAPEUTIC AGENT
FOR PANCREATIC DISEASE CONTAINING PANCREATIC BUD CELLS
Abstract
Provided is a method for generating pancreatic bud cells, having
the step of culturing PDX1.sup.+/NKX6.1.sup.+ cells in a medium
containing KGF, EGF and a BMP inhibitor. The culturing step may be
performed in suspension cultures or in adherent cultures. When the
cells are cultured in adherent cultures, the cells may be cultured
in a medium further containing a ROCK inhibitor or a nonmuscie
myosin II inhibitor.
Inventors: |
OSAFUNE; Kenji; (Kyoto,
JP) ; TOYODA; Taro; (Kyoto, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Kyoto University |
Kyoto |
|
JP |
|
|
Family ID: |
1000004500216 |
Appl. No.: |
16/589293 |
Filed: |
October 1, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15311759 |
Feb 14, 2017 |
10472610 |
|
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PCT/JP2015/064529 |
May 20, 2015 |
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16589293 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2501/117 20130101;
A61K 35/39 20130101; C12N 2501/155 20130101; C12N 2501/11 20130101;
C12N 2501/16 20130101; C12N 2501/727 20130101; C12N 2506/02
20130101; C12N 2501/41 20130101; C12N 5/0676 20130101; Y02A 20/402
20180101; C12N 2501/999 20130101 |
International
Class: |
C12N 5/071 20060101
C12N005/071; A61K 35/39 20060101 A61K035/39 |
Foreign Application Data
Date |
Code |
Application Number |
May 21, 2014 |
JP |
2014-105049 |
Claims
1. A method for generating pancreatic bud cells from pluripotent
stem cells, comprising the following steps: (1) culturing the
pluripotent stem cells in a medium containing an activin; (2)
culturing the cells obtained in step (1) in a medium containing
KGF; (3) culturing the cells obtained in step (2) in a medium
containing KGF, a BMP inhibitor, a retinoic acid derivative and a
hedgehog pathway inhibitor; and (4) dissociating the cells obtained
in step (3) into single cells and culturing the dissociated cells
in a medium containing KGF, a BMP inhibitor, and an agent selected
from the group consisting of ROCK inhibitor and nonmuscle myosin II
inhibitor, wherein the steps (1)-(4) are under an adherent culture
condition.
2. The method according to claim 1, wherein the ROCK inhibitor is
selected from the group consisting of Y-27632, Fasudil, SR3677,
GSK269962 and H-1152; or the nonmuscie myosin II inhibitor is
Blebbistatin.
3. The method according to claim 2, wherein the ROCK inhibitor is
Y-27632.
4. The method according to claim 3, wherein the concentration of
Y-27832 in the medium is 10-200 .mu.M.
5. The method according to claim 2, wherein the nonmuscie myosin II
inhibitor is Blebbistatin.
6. The method according to claim 5, wherein the concentration of
Blebbistatin in the medium is 5-20 .mu.M.
7. The method according to claim 1, wherein the medium containing
an activin in step (1) further contains a GSK3 inhibitor.
8. The method according to claim 7, wherein the GSK3 inhibitor is
CHIR93021.
9. The method according to claim 1, wherein the BMP inhibitor is
Noggin.
10. The method according to claim 1, wherein the retinoic acid
derivative is TTNPB.
11. The method according to claim wherein the hedgehog pathway
inhibitor is KAAD-cyclopamine.
12. The method according to claim 1, wherein the medium in step (4)
further comprises a TGF.beta. inhibitor.
13. The method according to claim 11, wherein the TGF.beta.
inhibitor is ALK5 inhibitor II.
14. The method according to claim 1, wherein the pancreatic bud
cells are PDX1.sup.+/NKX6.1.sup.+.
15. The method according to claim 1, wherein the pluripotent stem
cells are human cells.
16. A method for generating pancreatic bud cells from pluripotent
stem cells, comprising the following steps: (1) culturing the
pluripotent stem cells in a medium containing an activin and a GSK3
inhibitor; (2) culturing the cells obtained in step (I) in a medium
containing KGF; (3) culturing the cells obtained in step (2) in a
medium containing NSF, a BMP inhibitor, a retinoic acid derivative
and a hedgehog pathway inhibitor; and (4) dissociating the cells
obtained in step (3) into single cells and culturing the
dissociated cells in a medium containing KGF, a BMP inhibitor, a
TGF.beta. inhibitor and an agent selected from the group consisting
of ROCK inhibitor and nonmuscie myosin II inhibitor, wherein the
steps (1)-(4) are under an adherent culture condition.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/311,759, filed Feb. 14, 2017, which is a 35
U.S.C. .sctn. 371 filing of International Patent Application No.
PCT/JP2015/064529, filed May 20, 2015, which claims priority to
Japanese Patent Application No. 2014-105049, filed May 21, 2014,
the entire disclosures of which are hereby incorporated herein by
reference.
TECHNICAL FIELD
[0002] The present invention relates to a method for generating
pancreatic bud cells and a therapeutic agent for treating a
pancreatic disease containing the pancreatic bud cells generated by
the method. The present application further relates to a method for
treating a pancreatic disease by using the pancreatic bud
cells.
BACKGROUND ART
[0003] The pancreas functions as an exocrine gland which secretes
digestive enzymes such as pancreatic lipase, trypsin, elastase and
pancreatic amylase as well as an endocrine gland which secretes
pancreatic hormones such as glucagon, insulin, somatostatin and
pancreatic polypeptide (PP). Ghrelin has been known as a stomach
hormone and in recent years, it has been reported that ghrelin is
also secreted by cells in the pancreas. The pancreatic hormones are
produced by pancreas islets that are cell cluster composed of four
types of cells: a cells, .beta. cells, .delta. cells and PP cells
in the pancreas.
[0004] Insulin plays important roles in promoting glucose
utilization, protein synthesis and production and storage of
neutral fats, lowering blood glucose level as well as maintaining
blood glucose within a normal range. Glucagon is a hyperglycemic
hormone that functions via hepatic glycogenolysis or
gluconeogenesis, and plays an important role in regulating sugar
metabolism along with insulin. Somatostatin acts via a somatostatin
receptor and suppresses secretion of various hormones such as
glucagon and insulin in the pancreas. PP has been known as a
satiety factor that is secreted from the cells in the islets of
Langerhans in response to food intake, and suppresses the food
intake and suppresses body weight gain. Ghrelin stimulates food
intake and increases body weight by lowering fat oxidation.
[0005] Diabetes is a disease developed by insufficient secretion of
insulin or insufficient response to insulin. Once it is developed,
the disease is difficult to be cured completely. Diabetes is
roughly classified into two types: type 1 diabetes mellitus which
is an insulin-dependent diabetes, and type 2 diabetes mellitus
which is a non-insulin-dependent diabetes.
[0006] Type 2 diabetes mellitus is a chronic disease begins with
acquisition of insulin resistance, and is thought to be a type of
diabetes whose onset mechanisms involve lifestyle habits such as
obesity due to overeating or lack of exercise and stress. Type 2
diabetes mellitus is often developed in the middle-aged and elderly
people, and many of patients with diabetes are affected with type 2
diabetes mellitus.
[0007] On the other hand, type 1 diabetes mellitus is a disease
caused by destruction of .beta. cells (insulin-producing cells) by,
for example, autoimmune diseases and viral infections, which
obstruct secretion of insulin in the body. Type 1 diabetes mellitus
is mainly treated by a symptomatic therapy, i.e. by insulin
administration. Pancreas transplantation or pancreatic islet
transplantation is also employed so that the blood glucose level
which continuously fluctuates in the body of the patient can become
automatically controlled and the burden on the patients is reduced.
Pancreas or pancreatic islet transplantation can effectively
control the blood glucose level of the patient within the normal
range. However, sufficient number of the organs that can be used
for transplantation is not available. In addition, the patient who
received the transplantation needs to take immunosuppressants for
entire life to avoid immunorejection to a graft. Immunosuppressants
may cause problems of infections or side effects.
[0008] A strategy including inducing insulin-producing cell in
vitro from cells derived from a patient, and transplanting the
induced cells into the patient has been proposed and studied.
Insulin-producing cells can be obtained, for example, by obtaining
cells from the epithelium of the pancreatic duct of a patient and
differentiating the cells in vitro into insulin-producing
cells.
[0009] Insulin-producing cells may also be obtained from
pluripotent stem cells such as embryonic stem (ES) cells or induced
pluripotent stem (iPS) cells by inducing differentiation of the
cells into insulin-producing cells with an activin and retinoic
acid (RA) (Patent Literature 1, and Non-Patent Literatures 1 to 5).
Further, insulin-generating cells may also be generated by
introducing PDX1 into pluripotent stem cells and culturing the
cells (Patent Literature 2 and Patent Literature 3) as well as by
culturing the pluripotent stem cells in the presence of suitably
combined small molecule compounds (Patent Literature 4 and
Non-Patent Literature 6). However, it has not been reported that
any of the in vitro generated insulin-producing cells could
successfully developed glucose responsiveness in a living body. On
the other hand, it has been reported that pancreas precursor cells
were generated and the cells secreted insulin depending on the
glucose level when transplanted into a living body (Non-Patent
Literatures 7 and 8).
CITATION LIST
Patent Literature
[0010] Patent Literature 1: Japanese Patent Publication No.
2009-225661 [0011] Patent Literature 2: U.S. Pat. No. 7,534,608
[0012] Patent Literature 3: Japanese Patent Publication No.
2006-075022 [0013] Patent Literature 4: WO 2011/081222
Non-Patent Literature
[0013] [0014] Non-Patent Literature 1: E. Kroon et al., Nature
Biotechnology (2008) Vol. 26, No. 4: 443-452 [0015] Non-Patent
Literature 2: K. A. D'Amour et al., Nature Biotechnology (2006)
Vol. 24, No. 11: 1392-1401 [0016] Non-Patent Literature 3: W.
Jiang, Cell Research (2007) 17: 333-344 [0017] Non-Patent
Literature 4: J. H. Shim et al., Diabetologia (2007) 50: 1228-1238
[0018] Non-Patent Literature 5: R. Maehr et al., PNAS (2009), vol.
106, No. 37: 15768-15773 [0019] Non-Patent Literature 6: Kunisada Y
et al., Stem Cell Res. (2012) vol. 8, No. 2: 274-284. [0020]
Non-Patent Literature 7: Kroon E et al., Nat Biotechnol. (2008)
vol. 26, No. 4: 443-452. [0021] Non-Patent Literature 8: Rezania A
et al., Diabetes. (2012) vol. 61, No. 8: 2016-2029.
SUMMARY OF INVENTION
Technical Problem
[0022] In one aspect, an object of the present application is to
provide a method for inducing differentiation from
PDX1.sup.+/NKX6.1.sup.+ cells into pancreatic bud cells. More
specifically, the object is to provide the method further including
the step of inducing differentiation from pluripotent stem cells
into PDX1.sup.+/NKX6.1.sup.+ cells and then, into pancreatic bud
cells.
[0023] In another aspect, an object of the present application is
to provide a therapeutic agent for treating a pancreatic disease as
well as to provide a method for treating a pancreatic disease.
Solution to Problem
[0024] The inventors have conducted studies in earnest and have
found, for the first time, that pancreatic bud cells can be
obtained by inducing differentiation of PDX1.sup.+/NKX6.1.sup.+
cells by culturing the cells under a condition which causes the
formation of cellular aggregates in a medium containing KGF, EGF
and a BMP inhibitor. The present invention has been completed based
on such findings as above.
[0025] Namely, the present invention has the following features:
[0026] [1] A method for generating pancreatic bud cells, comprising
the step of culturing PDX1.sup.+/NKX6.1.sup.+ cells in a medium
containing KGF, EGF and a BMP inhibitor. [0027] [2] The method
according to [1], wherein the medium further contains a ROCK
inhibitor or a nonmuscle myosin II inhibitor. [0028] [3] The method
according to [2], wherein the ROCK inhibitor or the nonmuscle
myosin II inhibitor is a compound selected from the group
consisting of Y-27632, Fasudil, SR3677, GSK269962, H-1152 and
Blebbistatin. [0029] [4] The method according to [2] or [3],
wherein the cells are cultured under adherent culture conditions.
[0030] [5] The method according to [1], wherein the cells are
cultured under a condition which causes formation of cellular
aggregates. [0031] [6] The method according to any one of [1] to
[5], wherein the PDX1.sup.+/NKX6.1.sup.+ cells are generated from
pluripotent stem cells by a method comprising the following steps:
[0032] (1) culturing the pluripotent stem cells in a medium
containing an activin; and [0033] (2) culturing the cells obtained
in step (1) in a medium containing KGF. [0034] [7] The method
according to [6], wherein the medium containing an activin used in
step (1) further contains a GSK3 inhibitor. [0035] [8] The method
according to [6] or [7], wherein the medium containing KGF used in
step (2) further contains a BMP inhibitor, a retinoic acid
derivative and a hedgehog pathway inhibitor. [0036] [9] The method
according to any one of [1] to [8], wherein the BMP inhibitor is
Noggin. [0037] [10] The method according to any one of [7] to [9],
wherein the GSK3 inhibitor is CHIR99021. [0038] [11] The method
according to any one of [8] to [10], wherein the retinoic acid
derivative is TTNPB. [0039] [12] The method according to any one of
[8] to [11], wherein the hedgehog pathway inhibitor is
KAAD-cyclopamine. [0040] [13] The method according to any one of
[1] to [12], wherein the pancreatic bud cells are
PDX1.sup.+/NKX6.1.sup.+. [0041] [14] The method according to any
one of [1] to [13], wherein the pancreatic bud cells are human
cells. [0042] [15] A method for generating pancreatic bud cells
from pluripotent stem cells, comprising the following steps (i) to
(iii): [0043] (i) culturing the pluripotent stem cells in a medium
containing an activin; [0044] (ii) culturing the cells obtained in
step (i) in a medium containing KGF; and [0045] (iii) dissociating
the cells obtained in step (ii) into single cells and culturing the
cells in a medium containing KGF, EGF and a BMP inhibitor. [0046]
[16] The method according to [15], wherein the medium used in step
(iii) further contains a ROCK inhibitor or a nonmuscle myosin II
inhibitor. [0047] [17] The method according to [16], wherein the
ROCK inhibitor or the nonmuscle myosin II inhibitor is a compound
selected from the group consisting of Y-27632, Fasudil, SR3677,
GSK269962, H-1152 and Blebbistatin. [0048] [18] The method
according to [16] or [17], wherein in step (iii), the cells
obtained in step (ii) are dissociated into single cells, the
dissociated cells are cultured in a medium containing KGF, a BMP
inhibitor, a retinoic acid derivative and a hedgehog pathway
inhibitor, and then cultured in the medium containing KGF, EGF and
a BMP inhibitor. [0049] [19] The method according to any one of
[16] to [18], wherein in step (iii), the cells are cultured under
adherent culture conditions. [0050] [20] The method according to
[15], wherein in step (iii), the cells are cultured under
suspension culture conditions. [0051] [21] The method according to
any one of [15] to [20], wherein the medium containing an activin
used in step (i) further contains a GSK3 inhibitor. [0052] [22] The
method according to any one of [15] to [21], wherein the medium
containing KGF used in step (ii) further contains a BMP inhibitor,
a retinoic acid derivative and a hedgehog pathway inhibitor. [0053]
[23] The method according to any one of [15] to [22], wherein the
BMP inhibitor is Noggin. [0054] [24] The method according to any
one of [21] to [23], wherein the GSK3 inhibitor is CHIR99021.
[0055] [25] The method according to any one of [18] and [22] to
[24], wherein the retinoic acid derivative is TTNPB. [0056] [26]
The method according to any one of [18] and [22] to [25], wherein
the hedgehog pathway inhibitor is KAAD-cyclopamine. [0057] [27] The
method according to any one of [15] to [26], wherein the pancreatic
bud cells are PDX1.sup.+/NKX6.1.sup.+. [0058] [28] The method
according to any one of [15] to [27], wherein the pancreatic bud
cells are human cells. [0059] [29] A therapeutic agent for a
pancreatic disease, which comprises the pancreatic bud cells
generated by the method according to any one of [1] to [28]. [0060]
[30] The therapeutic agent according to [29], wherein the
pancreatic disease is diabetes. [0061] [31] The therapeutic agent
according to [30], wherein the diabetes is type 1 diabetes
mellitus. [0062] [32] Use of the pancreatic bud cells generated by
the method according to any one of [1] to [28], for the manufacture
of a therapeutic agent for treating a pancreatic disease. [0063]
[33] Pancreatic bud cells generated by the method according to any
one of [1] to [28], used for treating a pancreatic disease. [0064]
[34] A method for treating a pancreatic disease, which comprises
implanting the pancreatic bud cells generated by the method
according to any one of [1] to [28] to a subject in need of the
treatment of the pancreatic disease.
ADVANTAGEOUS EFFECTS OF INVENTION
[0065] The present inventors have made it possible to produce
pancreatic bud cells from PDX1.sup.+/NKX6.1.sup.+ cells, and have
found for the first time that the in vivo transplanted thus
produced pancreatic bud cells develop into insulin-producing cells
having glucose responsiveness. The pancreatic bud cells produced by
the method provided by the present application can be used for
regenerative therapies for pancreatic diseases such as
diabetes.
BRIEF DESCRIPTION OF DRAWINGS
[0066] FIG. 1 shows the summary of the protocol for generating
pancreatic bud cells from pluripotent stem cells.
[0067] FIG. 2 shows the ratio of PDX1.sup.+/NKX6.1.sup.+ cells and
PDX1.sup.+/NKX6.1.sup.+ cells in the cells obtained by each of the
induction procedures. In the figure, "d0" represents cells at the
end of Stage 2, "W/O" represents cells on Day 4 which were not
subjected to decomposition but were only subjected to exchange of
medium during Stage 3, "Monolayer" represents cells on Day 4 which
were subjected to adherent culture during Stage 3 and "Aggregate"
represents cells on Day 4 which were subjected to suspension
cultures during Stage 3. (N=3-4)
[0068] FIG. 3 shows the ratio of PDX1.sup.+/NKX6.1.sup.+ cells in
the cells differentiated from each of pluripotent stem cell lines
(KhES3, 585A1, 604B1, 692D2, 648B1 and 409B2). In the figure, "2D"
represents the case where adherent culture was performed during
Stage 3, and "Agg" represents the case where suspension culture was
performed during Stage 3. (Mean.+-.S.D., n=3)
[0069] FIG. 4A shows expression intensities of PDX1 and NKX6.1 in
the cells measured by using a flow cytometer at each of days 0, 1,
2, 4 and 12 of Stage 3. FIG. 4B shows the ratio of
PDX1.sup.+/NKX6.1.sup.+ cells at each of days 0, 4, 8, 12, 16 and
20 of Stage 3. (Mean.+-.S.D., n=3)
[0070] FIG. 5A shows cellular aggregates at Day 4 of Stage 3,
immunohistochemically stained with pancreatic bud cell makers,
PDX1, NKX6.1, SOX9 and GATA4.
[0071] FIG. 5B shows cellular aggregates at Day 4 of
[0072] Stage 3, immunohistochemically stained with endocrine cell
markers, INS, GCG, Somatostatin and Ghrelin.
[0073] FIG. 6A shows cellular aggregates at Day 12 of Stage 3, the
aggregates were immunohistochemically stained with pancreatic bud
cell markers, PDX1 , NKX6.1, SOX9 and GATA4.
[0074] FIG. 6B shows cellular aggregates at Day 12 of Stage 3
immunohistochemically stained with endocrine cell markers, INS,
GCG, Somatostatin and Ghreline.
[0075] FIG. 7A shows the shows the summary of the protocol for
determining culture conditions during Stage 3. In the figure, black
arrows indicate conditions under which Noggin (NOG), KGF and EGF
are added to the culture medium and white arrows indicate
conditions under which Noggin (NOG) is not added to the culture
medium or conditions under which none of Noggin (NOG), KGF and EGF
is added to the culture medium. FIG. 7B shows the ratio of
PDX1.sup.+/NKX6.1.sup.+ cells and PDX1.sup.+/NKX6.1.sup.+ cells in
the cells obtained under each condition.
[0076] FIG. 8A shows the shows the summary of the protocol for
determining effects of ALK5 inhibitor II during Stage 3. In the
figure, arrows indicate time periods when ALK5 inhibitor II was
added to the medium. FIG. 8B shows expressions of PDX1 (vertical
axes) and NKX6.1 (horizontal axes) under the condition where ALK5
inhibitor II was not added (the left hand in the figure) as well as
under the condition where ALK5 inhibitor II was added (the right
hand in the figure). The expressions were measured by a flow
cytometer. In the figure, numbers indicate the ratio of PDX1.sup.+
and NKX6.1.sup.+ cells contained in the culture.
[0077] FIG. 9 shows the epididymal fat pads of an immunodeficient
mouse (NOD. CB17-Prkdc.sup.scid/J) at 30 days after the induced
pancreatic bud cells were implanted. The place where the cells were
implanted was immunohistochemically stained with PDX and
insulin.
[0078] FIG. 10A shows plasma human C-Peptide levels in
immunodeficient mice that received kidney subcapsular implantation
of the induced pancreatic bud cells. The results are plotted to
days after implantation. FIG. 10B shows the change of plasma human
C-Peptide levels caused by glucose challenges (+) in mice to which
the induced pancreatic bud cells were implanted.
[0079] FIG. 11A shows the shows the summary of the protocol for
generating pancreatic bud cells from pluripotent stem cells under
adhesive conditions.
[0080] FIG. 11B shows cells obtained by culturing in the presence
of 50 .mu.M of Y-27632 during Stage 3 (right) and
immunohistochemically stained with pancreatic bud cell markers,
PDX1 (lower) and NKX6.1 (upper). In the figure, "Water" (left)
indicates the negative control. FIG. 11C shows the ratio between
PDX1.sup.+/NKX6.1.sup.+ cells and PDX1.sup.+/NKX6.1.sup.+ cells to
the concentration of Y-27632 during Stage 3.
[0081] FIG. 12A shows the shows the summary of the modified
protocol for generating pancreatic bud cells from pluripotent stem
cells under adhesive conditions.
[0082] FIG. 12B shows the cells induced by culturing in the
presence of 50 .mu.M of Fasudil during Stage 3 in the modified
protocol (right), and immunohistochemically stained with pancreatic
bud cell markers, PDX1 (lower) and NKX6.1 (upper). In the figure,
"Water" (left) indicates negative controls. FIG. 12C shows the
ratio between PDX1.sup.+/NKX6.1.sup.+ cells and
PDX1.sup.+/NKX6.1.sup.+ cells to the concentration of Fasudil
during Stage 3. In the figure, "Y50" indicates the ratio of the
cells obtained by culturing in the presence of 50 .mu.M of Y-27632
during Stage 3 and is a positive control.
[0083] FIG. 13A shows cells obtained by culturing in the presence
of 5 .mu.M of SR3677 during Stage 3 in the modified protocol and
immunohistochemically stained with NKX6.1. In the figure, "DMSO"
(left) indicates a negative control. FIG. 13B shows the ratio of
NKX6.1.sup.+ cells in relation to the concentration of SR3677
during Stage 3 in the modified protocol. In the figure, "Y50"
indicates the ratio of the cells obtained by culturing in the
presence of 50 .mu.M of Y-27632 during Stage 3 and is a positive
control.
[0084] FIG. 13C shows the cells obtained by culturing in the
presence of 0.1 .mu.M of GSK269962 during Stage 3 in the modified
protocol and immunohistochemically stained with NKX6.1 (right). In
the figure, "DMSO" (left) indicates a negative control. FIG. 13D
shows the ratio of NKX6.1.sup.+ cells obtained with each
concentration of GSK269962 during Stage 3 in the modified protocol.
In the figure, "Y50" indicates the ratio of the cells obtained by
culturing in the presence of 50 .mu.M of Y-27632 during Stag 3 in
the modified protocol and is a positive control.
[0085] FIG. 14A shows the cells obtained by culturing in the
presence of 50 .mu.M of H-1152 (right) during Stage 3 in the
modified protocol and immunohistochemically stained with NKX6.1. In
the figure, "DMSO" (left) indicates a negative control. FIG. 14B
shows the ratio of NKX6.1.sup.+ cells to the concentration of
H-1152 during Stage 3 in the modified protocol. In the figure,
"Y50" indicates the ratio of the cells obtained by culturing in the
presence of 50 .mu.M of Y-27632 during Stag 3 in the modified
protocol and is a positive control.
[0086] FIG. 14C shows the cells obtained by culturing in the
presence of 5 .mu.M of Blebbistatin (right) during Stage 3 in the
modified protocol and immunohistochemically stained with pancreatic
bud cell markers, PDX1 (lower) and NKX6.1 (upper). In the figure,
"DMSO" (left) indicates a negative control. FIG. 14D shows the
ratio of PDX1.sup.+/NKX6.1.sup.+ cells to concentration of
Blebbistatin during Stage 3 in the modified protocol. In the
figure, "Y50" indicates the ratio of the cells obtained by
culturing in the presence of 50 .mu.M of Y-27632 during Stag 3 in
the modified protocol and is a positive control.
DESCRIPTION OF EMBODIMENTS
[0087] The present application provides, in one aspect, a method
for generating pancreatic bud cells, comprising the step of
culturing PDX1.sup.+/NKX6.1.sup.+ cells in a medium containing KGF,
EGF and a BMP inhibitor.
[0088] Pancreatic bud cells refer to cells which are capable of
differentiating into cells which constitute the pancreas such as
endocrine cells, pancreatic duct cells and exocrine cells. Examples
of pancreatic bud cells include cells which express at least PDX1
and NKX6.1. The pancreatic bud cells may also express genetic
markers such as SOX9 and GATA4.
[0089] The pancreatic bud cells generated according to this aspect
may be provided as a cell population which also contains cells
other than pancreatic bud cells, or as a purified cell population.
Preferably, the cell population contains pancreatic bud cells by
30% or more, 40% or more, 50% or more, 60% or more, 70% or more, or
80% or more.
[0090] For generating pancreatic bud cells from
PDX1.sup.+/NKX6.1.sup.+ cells, the PDX1.sup.+/NKX6.1.sup.+ cells
may be cultured in a medium containing KGF, EGF and a BMP inhibitor
under a condition which causes formation of cellular aggregates.
The phrase "condition which causes formation of cellular
aggregates" refers any of cell culture conditions which provide
cellular aggregates in the medium. For example, cells may be
substantially dissociated or decomposed from adherent culture into
single cells by a conventional procedure and then, cultured in
suspension cultures. Alternatively, previously generated cellular
aggregates may be cultured. Preferably, cells are cultured in
suspension cultures after being dissociated into single cells. By
culturing dissociated single cells in suspension cultures, cells
adhere to each other to form cellular aggregates. Cellular
aggregates may also be formed by centrifuging the dissociated
cells. Cells may be dissociated by, for example, a mechanical
dissociation, or by using a dissociation solution such as a
solution having protease and collagenase activities. Examples of
dissociation solutions may include those containing trypsin, or
trypsin and collagenase, Accutase.TM. and Accumax.TM. (Innovative
Cell Technologies, Inc.)). In addition, a dissociation solution
having only collagenase activity may also be used.
[0091] Suspension cultures refer to cultures in which cells are
cultured in a state where the cells do not adhere to the culture
dish. Suspension cultures may be performed by using, but not
particularly limited to, a culture dish which has not been treated
to improve cellular adhesiveness (for example, extracellular matrix
coatings), or a culture dish which has been treated to suppress
cellular adhesion (for example, poly-hydroxyethyl methacrylate
(poly-HEMA) coating or a polymer of
2-methacryloyloxyethylphosphorylcholine (Lipidure) coating).
[0092] The size of a cellular aggregate is not particularly
limited, and a cellular aggregate may be formed by at least
3.times.10.sup.3 or more cells, and for example, by
1.times.10.sup.4 or more, 2.times.10.sup.4 or more,
3.times.10.sup.4 or more, 4.times.10.sup.4 or more and
5.times.10.sup.4 or more cells.
[0093] In another aspect, PDX1.sup.+/NKX6.1.sup.+ cells are
dissociated into single cells and cultured in adherent cultures.
The cells are dissociated into single cells and cultured in
adherent cultures in a medium used in Stage 2 described below. The
time period of adherent cultures in the medium used in Stage 2 is
not particularly limited, and may be one day or more, 2 days or
more, and three days or more. Preferable time period is one day.
The medium used in Stage 2 may comprise a ROCK inhibitor in order
to suppress apoptosis of the cells after aggregates of pluripotent
stem cells are dissociated into single cells. ROCK inhibitors
discussed below may be used in this embodiment and preferable ROCK
inhibitor is Y-27632.
[0094] In the specification and claims of the present application,
adherent cultures refer to any of culturing methods wherein culture
dish used for the culture is suitably coated. Examples of coating
materials include Matrigel (BD Biosciences), Synthemax (Corning),
gelatins, extracellular proteins, for example, collagen, laminin
such as laminin-111, -411 or -511, heparan sulfate proteoglycan and
entactin, fragments of the extracellular protein and combinations
thereof.
[0095] The medium used for culturing PDX1.sup.+/NKX6.1.sup.+ cells
to generate pancreatic bud cells can be prepared by adding KGF, EGF
and a BMP inhibitor appropriately to a basal medium for animal cell
culture. Examples of basal media include MEM Zinc Option, IMEM Zinc
Option, IMDM, Medium 199, Eagle's Minimum Essential Medium (EMEM),
.alpha.-MEM, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12
Medium, RPMI 1640 Medium, Fischer's Medium, and mixtures of these
media. The basal medium may be supplemented with serum, for
example, fetal bovine serum (FBS). Alternatively, be a serum-free
medium may be used. As required, the basal medium may contain, for
example, one or more alternatives to sera such as albumin,
transferrin, KnockOut Serum Replacement (KSR, an alternative to
serum used for culturing ES cells) (Invitrogen), N2 Supplement
(Invitrogen), B27 Supplement (Invitrogen), a fatty acid, insulin, a
collagen precursor, a trace element, 2-mercaptoethanol and
3'-Thioglycerol, and the basal medium may contain one or more
substances such as a lipid, an amino acid, L-glutamine, GlutaMAX
(Invitrogen), a nonessential amino acid (NEAA), a vitamin, a growth
factor, an antibiotic, an antioxidant, pyruvic acid, a buffer
agent, an inorganic salt, and equivalents thereof. In one
embodiment, the basal medium is IMEM Zinc Option containing B-27
Supplement.
[0096] KGF is a protein called Keratinocyte Growth Factor and is
sometimes also referred to as FGF-7. KGF is commercially available
from, for example, R&D systems, Inc. The concentration of KGF
may be from 1 ng/ml to 1 pg/ml, preferably from 5 ng/ml to 500
ng/ml, and more preferably from 10 ng/ml to 200 ng/ml.
[0097] EGF is a protein called epidermal growth factor. EGF is
commercially available from, for example, R&D systems, Inc. The
concentration of EGF may be from 1 ng/ml to 1 .mu.g/ml, preferably
from 5 ng/ml to 500 ng/ml, and more preferably from 10 ng/ml to 100
ng/ml.
[0098] Examples of the BMP inhibitors include protein inhibitors
such as Chordin, Noggin and Follistatin; Dorsomorphin or
6-[4-(2-piperidin-1-yl-ethoxy)phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a]pyrim-
idine and a derivative thereof (P. B. Yu et al. (2007),
Circulation, 116: II_60, P. B. Yu et al. (2008), Nat. Chem. Biol.,
4:33-41, J. Hao et al. (2008), PLoS ONE, 3 (8): e2904), and
LDN-193189 or
4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline.
Preferable BMP inhibitor is Noggin. Noggin is commercially
available, for example, from Peprotech.
[0099] When Noggin is used as BMP inhibitor, the concentration of
Noggin may be from 1 ng/ml to 1 .mu.g/ml, preferably from 5 ng/ml
to 500 ng/ml, and more preferably from 50 ng/ml to 200 ng/ml.
[0100] The medium used for culturing PDX1.sup.+/NKX6.1.sup.+ cells
to generate pancreatic bud cells may further contain a ROCK
inhibitor or a nonmuscle myosin II inhibitor. When the
above-described PDX1.sup.+/NKX6.1.sup.+ cells are cultured in
adherent cultures, it is preferable that the ROCK inhibitor or the
nonmuscle myosin II inhibitor is further added to the medium.
[0101] The ROCK inhibitor is not particularly limited as long as it
can suppress the function of Rho-kinase (ROCK), and examples of
ROCK inhibitor include Y-27632 (for example, see Ishizaki et al.,
Mol. Pharmacol. 57, 976-983 (2000); Narumiya et al., Methods
Enzymol. 325, 273-284 (2000)), Fasudil/HA1077 (Uenata et al.,
Nature 389: 990-994 (1997)), SR3677 (Feng Y et al., J Med Chem. 51:
6642-6645 (2008)), GSK269962 (Stavenger R A et al., J Med Chem. 50:
2-5 (2007) or WO 2005/037197), H-1152 (Sasaki et al., Pharmacol.
Ther. 93: 225-232 (2002)), Wf-536 (Nakajima et al., Cancer
Chemother Pharmacol. 52 (4): 319-324 (2003)) and derivatives
thereof, as well as an antisense nucleic acid against ROCK, an RNA
interference-inducing nucleic acid (for example, an siRNA) against
ROCK, a dominant negative mutant of ROCK, and expression vectors
for them. In addition, as the ROCK inhibitor, other known low
molecular weight compounds may be used (U.S. patent application
publications Nos. 2005/0209261, 2005/0192304, 2004/0014755,
2004/0002508, 2004/0002507, 2003/0125344 and 2003/0087919, as well
as International Publications Nos. WO 2003/062227, WO 2003/059913,
WO 2003/062225, WO 2002/076976, and WO 2004/039796). In the present
invention, one or more kinds of ROCK inhibitors may be used.
Examples of the ROCK inhibitors which are preferably used in this
step include Y-27632, Fasudil/HA1077, SR3677, GSK269962 and
H-1152.
[0102] When Y-27632 is used as the ROCK inhibitor, the
concentration of Y-27632 in the medium may be from 0.1 .mu.M to 100
.mu.M, preferably from 1 .mu.M to 500 .mu.M, and more preferably
from 10 .mu.M to 200 .mu.M.
[0103] When Fasudil/HA1077 is used as the ROCK inhibitor, the
concentration of Fasudil/HA1077 in the medium may be from 1 .mu.M
to 100 .mu.M, and preferably from 10 .mu.M to 100 .mu.M.
[0104] When SR3677 is used as the ROCK inhibitor, the concentration
of SR3677 in the medium may be from 0.1 .mu.M to 50 .mu.M, and
preferably from 0.5 .mu.M to 50 .mu.M.
[0105] When GSK269962 is used as the ROCK inhibitor, the
concentration of GSK269962 in the medium may be from 0.001 .mu.M to
100 .mu.M, is preferably from 0.005 .mu.M to 50 .mu.M, and more
preferably from 0.05 .mu.M to 120 .mu.M.
[0106] When H-1152 is used as the ROCK inhibitor, the concentration
of H-1152 in the medium may be from 5 .mu.M to 100 .mu.M, and
preferably from 10 .mu.M to 50 .mu.M.
[0107] The nonmuscle myosin II inhibitor may be an ATPase inhibitor
that inhibits the ATPase activity of the heavy chain subunit of
nonmuscle myosin IIA, nonmuscle myosin IIB which is one of heavy
chain isoforms of nonmuscle myosin II or a myosin light chain
kinase inhibitor. Examples of the nonmuscle myosin II inhibitors
may include blebbistatin (Blebbistatin) A3, Calphostin C, Goe6976,
Goe7874, Fasudil/HA1077, Hypericin, K-252a, KT5823, ML-7, ML-9,
Piceatannol, Staurosporine, W-5, W-7, W-12, W-13 and
Wortmannin.
[0108] Preferred nonmuscle myosin II inhibitors are blebbistatin
and Fasudil/HA1077.
[0109] When blebbistatin is used as the nonmuscle myosin II
inhibitor, the concentration of blebbistatin in the medium may be
from 1 .mu.M to 200 .mu.M, and preferably from 10 .mu.M to 100
.mu.M.
[0110] The medium used for the step of culturing
PDX1.sup.+/NKX6.1.sup.+ cells to generate pancreatic bud cells
according to the present invention may further contain a TGF.beta.
inhibitor. The TGF.beta. inhibitor is a substance which inhibits
the signal transduction starts from the binding of TGF.beta. to its
receptor and leads to SMAD. The TGF.beta. inhibitor is not
particularly limited as long as it inhibits the binding of
TGF.beta. to the receptor, an ALK family protein, or inhibits
phosphorylation of SMAD caused by the ALK family protein. Examples
of TGF.beta. inhibitors include Lefty-1 (NCBI Accession Nos:
NM_010094 (mouse), and NM_020997(human)), SB431542 and SB202190 (R.
K. Lindemann et al., Mol. Cancer, 2003, 2: 20), SB505124
(GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios),
LY2109761, LY364947, LY580276 (Lilly Research Laboratories),
A-83-01 (WO 2009/146408), ALK5 inhibitor II
(2-[3-[6-methylpyridin-2-yl]-1H-pyrazol-4-yl]-1,5-naphthyridine),
TGF.beta.RI kinase inhibitor VIII
(6-[2-tert-butyl-5-[6-methyl-pyridin-2-yl]-1H-imidazol-4-yl]-quinoxaline)
and derivatives thereof. Preferably, the TGF.beta. inhibitor may be
ALK5 inhibitor II.
[0111] When ALK5 inhibitor II is used as the TGF.beta. inhibitor,
the concentration of ALK5 inhibitor II in the medium may be from
0.01 .mu.M to 100 .mu.M, is preferably from 0.1 .mu.M to 50 .mu.M,
and more preferably from 1 .mu.M to 20 .mu.M.
[0112] The TGF.beta. inhibitor may preferably be added two days
after the start of culturing PDX1.sup.+/NKX6.1.sup.+ cells. The
time period of addition of the TGF.beta. inhibitor is not
particularly limited, and may be one day or more, 2 days or more, 3
days or more, or 4 days or more.
[0113] The maximum time period of the Stage of culturing
PDX1.sup.+/NKX6.1.sup.+ cells to generate pancreatic bud cells is
not limited since culturing for a long period of time does not
particularly affect the generation efficiency of pancreatic bud
cells. For example, the cells in this Stage may be cultured for 4
days or more, 5 days or more, 6 days or more, 7 days or more, 8
days or more, days or more, 10 days or more, 11 days or more, 12
days or more, 13 days or more, or 14 days or more. Preferably, the
cells may be cultured in this Stage for 4 days or more and 20 days
or less.
[0114] In the Stage of culturing PDX1.sup.+/NKX6.1.sup.+ cells to
generate pancreatic bud cells, the cells may be cultured at a
temperature from about 30.degree. C. to about 40.degree. C., and
preferably about 37.degree. C. In this step, the cells are cultured
under a CO.sub.2-containing air atmosphere, and the concentration
of CO.sub.2 is preferably from about 2% to about 5%.
[0115] PDX1.sup.+/NKX6.1.sup.+ cells are not particularly limited
as long as the cells express PDX1 but do not express NKX6.1. The
phrase "cells express PDX1 " means that PDX1 gene or PDX1 gene
product can be detected in the cells by a known method, and the
phrase "cells do not express NKX6.1" means that NKX6.1 gene or
NKX6.1 gene product cannot be detected by any known method.
Examples of the known detection method include immunostaining.
[0116] PDX1.sup.+/NKX6.1.sup.+ cells may be isolated from the
living body or may be generated from other types of cells such as
pluripotent stem cells by a known method. In a certain embodiment,
PDX1.sup.+/NKX6.1.sup.+ cells may be those generated from
pluripotent stem cells by a method comprising the following stages:
[0117] (Stage 1) culturing the pluripotent stem cells in a medium
containing an activin; and [0118] (Stage 2) culturing the cells
obtained in Stage 1 in a medium containing KGF.
[0119] In one embodiment, a method for generating pancreatic bud
cells from pluripotent stem cells including the stage of inducing
pancreatic bud cells from PDX1.sup.+/NKX6.1.sup.+ cells as Stage 3
of the above-described method is provided: [0120] (Stage 1)
culturing the pluripotent stem cells in a medium containing an
activin; [0121] (Stage 2) culturing the cells obtained in Stage 1
in a medium containing KGF; and [0122] (Stage 3) dissociating the
cells (PDX1.sup.+/NKX6.1.sup.+ cells) obtained in Stage 2 into
single cells and culturing the cells in a medium containing KGF,
EGF and a BMP inhibitor.
[0123] The medium used for culturing the pluripotent stem cells in
a medium containing an activin (Stage 1) according to these
embodiments can be prepared by adding an activin a basal medium for
animal cells. Examples of the basal media include MEM Zinc Option,
IMEM Zinc Option, IMDM, Medium 199, Eagle's Minimum Essential
Medium (EMEM), .alpha.-MEM, Dulbecco's modified Eagle's Medium
(DMEM), Ham's F12 Medium, RPMI 1640 Medium, Fischer's Medium, and
mixtures of these media. The basal medium may be supplemented with
serum, for example, fetal bovine serum (FBS), or the basal medium
may be a serum-free medium. As required, the basal medium may
contain, for example, one or more alternatives to sera such as
albumin, transferrin, KnockOut Serum Replacement (KSR) (an
alternative to serum used for culturing ES cells) (Invitrogen), N2
Supplement (Invitrogen), B-27 Supplement (Invitrogen), a fatty
acid, insulin, a collagen precursor, a trace element,
2-mercaptoethanol and 3'-Thioglycerol, and the basal medium may
contain one or more substances such as a lipid, an amino acid,
L-glutamine, GlutaMAX (Invitrogen), a nonessential amino acid
(NEAA), a vitamin, a growth factor, an antibiotic, an antioxidant,
pyruvic acid, a buffer agent, an inorganic salt, and equivalents
thereof. In one embodiment, the basal medium is RPMI 1640 medium
containing B-27 Supplement.
[0124] During Stage 1 in this embodiment, single cell suspension of
pluripotent stem cells may be prepared by substantially
dissociating or decomposing the cellular aggregates by a
conventional procedure and cultured. Alternatively, cellular
aggregates of pluripotent stem cells in which cells adhere each
other may be cultured. Preferably, suspension culture of single
cells may be employed. Examples of the procedures for dissociating
cells include mechanical dissociation, chemical dissociation using
a dissociation solution such as a solution having both protease and
collagenase activities such as a solution containing trypsin and
collagenase, Accutase.TM., Accumax.TM. (Innovative Cell
Technologies, Inc.)) or a solution having only collagenase
activity. Pluripotent stem cells may be cultured in adherent
cultures by using a coated culture dish.
[0125] Adherent cultures during Stage 1 of this embodiment can be
performed under the same conditions as those described above.
[0126] As an activin, any activins including activin A, activin B,
activin C, activin D and activin AB may be used, and activin A is
preferable. In addition, as an activin, any one of activins derived
from mammals such as human and mouse may be used. As the activin
used for the present invention, an activin derived from the same
animal as the animal from which pluripotent stem cells used for
differentiation are derived is preferably used, and for example,
when pluripotent stem cells derived from human are used as starting
materials, an activin derived from human is preferably used. These
activins are commercially available.
[0127] The concentration of the activin in the medium may be from
0.1 to 200 ng/ml, preferably from 5 to 150 ng/ml, and more
preferably from 10 to 100 ng/ml.
[0128] The medium used in Stage 1 may further contain a GSK3
inhibitor and/or a ROCK inhibitor. A GSK3 inhibitor is defined as a
substance which inhibits the kinase activity of GSK-3.beta. protein
such as an ability to phosphorylate .beta.-catenin, and many GSK3
inhibitors are known. Examples of the GSK3 inhibitors include BIO
(also called GSK-3.beta. inhibitor IX; 6-bromoindirubin3'-oxime)
which is a derivative of indirubin, 5B216763
(3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione)
and 5B415286
(3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dio-
ne) which are derivatives of maleimide, GSK-3.beta. inhibitor VII
(4-dibromoacetophenone) which is a phenyl a bromomethylketone
compound, L803-mts (also called, GSK-3.beta. peptide inhibitor;
Myr-N-GKEAPPAPPQSpP-NH2) which is a cell-penetrating phosphorylated
peptide and CHIR99021
(6-[2-[4-(2,4-Dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-yl-
amino]ethylamino]pyridine-3-carbonitrile) which has a high
selectivity. These compounds are commercially available, for
example, from Calbiochem and Biomol. These compounds may be
obtained from other suppliers or may be prepared by the user
himself. The GSK-3.beta. inhibitor used for the present invention
may preferably be CHIR99021.
[0129] When CHIR99021 is used, the concentration of CHIR99021 in
the medium may be from 0.01 .mu.M to 100 .mu.M, preferably from 0.1
.mu.M to 10 .mu.M, and more preferably from 1 .mu.M to 5 .mu.M.
[0130] The GSK3 inhibitor may preferably be added at the start of
culturing pluripotent stem cells. The time period for culturing the
cells in the presence of the GSK3 inhibitor is not particularly
limited, and may be day or more, 2 days or more, or 3 days or more.
Preferably, the time period is from one day to three days.
[0131] The ROCK inhibitor used in Stage 1 may be the same as one of
the above-described ROCK inhibitors and may preferably be Y-27632.
The ROCK inhibitor can be used in order to suppress apoptosis of
the cells after the pluripotent stem cells are dissociated into
single cells. The time period for culturing the cells in the
presence of the ROCK inhibitor is not particularly limited, and may
be 1 day or more, or 2 days or more, and preferably, 1 day.
[0132] There is no particular upper limit of the period of Stage 1.
Longer period of culture does not particularly affect the
efficiency for generating pancreatic bud cells. The period of Stage
1 may be 3 days or more, 4 days or more, 5 days or more, 6 days or
more, or 7 days or more. Preferably, Stage 1 may be 4 days.
[0133] In Stage 2, the medium used for culturing the cells obtained
in Stage 1 may be prepared by adding KGF to a basal medium for
animal cells. Examples of the basal media may include MEM Zinc
Option, IMEM Zinc Option, IMDM, Medium 199, Eagle's Minimum
Essential Medium (EMEM), .alpha.-MEM, Dulbecco's modified Eagle's
Medium (DMEM), Ham's F12 Medium, RPMI 1640 Medium, Fischer's
Medium, and mixtures of these media. The basal medium may contain
serum (for example, fetal bovine serum (FBS)) or the basal medium
may be a serum-free medium. As required, the basal medium may
contain, for example, one or more alternatives to sera such as
albumin, transferrin, KnockOut Serum Replacement (KSR) (an
alternative to serum used for culturing ES cells) (Invitrogen), N2
Supplement (Invitrogen), B-27 Supplement (Invitrogen), a fatty
acid, insulin, a collagen precursor, a trace element,
2-mercaptoethanol and 3'-Thioglycerol, and the basal medium may
contain one or more substances such as a lipid, an amino acid,
L-glutamine, GlutaMAX (Invitrogen), a nonessential amino acid
(NEAA), a vitamin, a growth factor, an antibiotic, an antioxidant,
pyruvic acid, a buffer agent, an inorganic salt, and equivalents
thereof. In one embodiment, the basal medium is IMEM Zinc Option
containing B-27 Supplement.
[0134] KGF used in Stage 2 may be the same as the above-described
KGF. The concentration of KGF in Stage 2 may preferably be lower
than the above-described concentration, and for example, the
concentration of KGF in Stage 2 may be from 1 ng/ml to 500 ng/ml,
and preferably from 10 ng/ml to 100 ng/ml.
[0135] The medium used in Stage 2 may further contain a BMP
inhibitor, a retinoic acid derivative and a hedgehog pathway
inhibitor.
[0136] The BMP inhibitor used in Stage 2 may be used under the same
conditions to those described above.
[0137] The retinoic acid derivative used in Stage 2 covers an
artificially modified retinoic acid which retains the functions of
natural retinoic acid, and examples may include
4-[[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbonyl]amino-
]-Benzoic acid (AM580) (Tamura K, et al., Cell Differ. Dev. 32:
17-26 (1990)),
4-[(1E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl-
)-1-propen-1-yl]-Benzoic acid (TTNPB) (Strickland S, et al., Cancer
Res. 43: 5268-5272 (1983)), retinol palmitate, retinol, retinal,
3-dehydroretinoic acid, 3-dehydroretinol, 3-dehydroretinal, and
compounds described in Abe, E., et al., Proc. Natl. Acad. Sci.
(USA) 78: 4990-4994 (1981), Schwartz, E. L. et al., Proc. Am.
Assoc. Cancer Res. 24: 18 (1983), and Tanenaga, K. et al., Cancer
Res. 40: 914-919 (1980). Examples of the retinoic acid derivative
preferably used in this Stage include TTNPB. The concentration of
the retinoic acid derivative used in this Stage can be
appropriately selected by those skilled in the art depending on the
retinoic acid derivative used. When TTNPB is used, its
concentration in the medium may be from 1 nM to 100 nM, preferably
from 5 nM to 50 nM, and more preferably from 5 nM to 10 nM.
[0138] The hedgehog pathway inhibitor used in Stage 2 means a
substance which inhibits signals, such as Smoothened, elicited by
the binding of any one of Sonic Hedgehog, Indian hedgehog and
Desert Hedgehog to their membrane receptor, Patched. The hedgehog
pathway inhibitor may be any substance that inhibits signals
elicited by the binding of a Hedgehog to its receptor. Examples of
the hedgehog pathway inhibitor include cyclopamine, jervine,
3-Keto-N-(aminoethyl-aminocaproyl-dihydro-cinnamoyl) (KAAD
)-cyclopamine, CUR-61414, SANT-1, SANT-2, SANT-3, SANT-4, IPI-926,
IPI-269609, GDC-0449 and NVP-LDE-225. Preferably, the hedgehog
pathway inhibitor is KAAD-cyclopamine. The concentration of the
hedgehog pathway inhibitor used in Stage 2 can be appropriately
selected by those skilled in the art depending on the hedgehog
pathway inhibitor used. When KAAD-cyclopamine is used, its
concentration in the medium may be from 0.1 nM to 1 .mu.M, and
preferably from 1 nM to 500 nM.
[0139] In the specification and claims of the present application,
pluripotent stem cells refer to stem cells which have pluripotency,
that is the ability of cells to differentiate into all type of the
cells in the living body, as well as proliferative capacity.
Examples of the pluripotent stem cells include embryonic stem (ES)
cells (J. A. Thomson et al. (1998), Science 282: 1145-1147; J. A.
Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92: 7844-7848;
J. A. Thomson et al. (1996), Biol. Reprod., 55: 254-259; J. A.
Thomson and V. S. Marshall (1998), Curr. Top. Dev. Biol., 38:
133-165), nuclear transfer embryonic stem (ntES) cells that can be
obtained by nuclear transplantation into the ES cells (T. Wakayama
et al. (2001), Science, 292: 740-743; S. Wakayama et al. (2005),
Biol. Reprod., 72: 932-936; J. Byrne et al. (2007), Nature, 450:
497-502), germline stem cells ("GS cells") (M. Kanatsu-Shinohara et
al. (2003) Biol. Reprod., 69: 612-616; K. Shinohara et al. (2004),
Cell, 119: 1001-1012), embryonic germ cells ("EG cells") (Y. Matsui
et al. (1992), Cell, 70: 841-847; J. L. Resnick et al. (1992),
Nature, 359: 550-551), induced pluripotent stem (iPS) cells (K.
Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi
et al. (2007), Cell, 131:861-872; J. Yu et al. (2007), Science,
318: 1917-1920; Nakagawa, M. et al., Nat. Biotechnol. 26: 101-106
(2008); WO 2007/069666), pluripotent cells derived from cultured
fibroblasts and bone marrow stem cells (Multi-lineage
differentiating Stress Enduring cells, Muse cells) (WO
2011/007900). More preferably, the pluripotent stem cells are human
pluripotent stem cells.
[0140] The iPS cells are particularly preferable as pluripotent
stem cells for generating pancreatic bud cells used for
implantation. For therapeutic use, it is desirable that the iPS
cells are those induced from somatic cells of an individual who has
the same or substantially the same HLA genotypes as those of the
individual who receives implantation of the cells, from the
viewpoint that the cells do not cause rejection reaction. In this
regard, the phrase "substantially the same HLA" means HLAs between
the donner and the recipient match to a degree that the
immunoreaction against the implanted cells can be suppressed by an
immunosuppressant. For example, substantially the same HLA may
cover three genes match, i.e. HLA-A, HLA-B, and HLA-DR match or
four genes match, i.e. HLA-A, HLA-B, HLA-DR and HLA-C mach.
[0141] In another embodiment, pancreatic bud cells obtained through
the above-described procedures may be used as a medicament, in
particular, for cellular therapy. The cells may be, before being
administered, irradiated with radiation or treated with a compound
which suppresses proliferation of the cells such as mitomycin
C.
[0142] The pancreatic bud cells may be suspended in physiological
saline or the like and the suspension may be administered directly
to the pancreas, the mesenterium, the spleen, the liver or the
kidney, in particular into the kidney subcapsule of the patient.
Alternatively, the cells may be encapsulated with polyvinyl alcohol
(PVA) (Qi Z et al., Cell Transplant. 21: 525-534 (2012)) or alginic
acid (Dufrane D, et al., Transplantation 90: 1054-1062 (2010)) and
the capsule may be administered. The cells may be administered in
combination with a scaffolding material such as polyethylene
glycol, gelatin, or collagen. The number of the cells administered
may be appropriately determined depending on the body size, and may
be, for example, from 1.times.10.sup.8 to 1.times.10.sup.10
cells/body, is preferably from 5.times.10.sup.8 to
1.times.10.sup.10 cells/body, and more preferably from
1.times.10.sup.9 to 1.times.10.sup.10 cells/body.
[0143] The pancreatic bud cells may be used for treating a
pancreatic disease. Examples of the pancreatic diseases include
acute pancreatitis, chronic pancreatitis, diabetes, pancreatic
cancer and islet of Langerhans tumor. The pancreatic bud cells
according to the present invention are induced to be
insulin-producing cells which secrete insulin in response to
glucose level in the body and are effective for treating diabetes.
In particular, the medicament containing the pancreatic bud cells
are effective for treating type 1 diabetes mellitus in which
insulin-producing cells die.
[0144] The origin of the cells described in the specification are
not particularly limited and may include human and nonhuman
animals, for example, mice, rats, cattle, horses, pigs, sheep,
monkeys, dogs, cats and birds. Human cells are preferably used.
[0145] The present invention is described in more detail referring
to following Examples. The present invention, however, is not
limited by those Examples in any way.
EXAMPLE 1
[0146] Induction of differentiation into pancreatic bud cells
[0147] Human ES cell line KhES3 gifted from Kyoto University was
used. Cells were cultured according to a conventional procedure (H.
Suemori et al. (2006), Biochem. Biophys. Res. Commun.,
345:926-932). (Alternatively, the cells were cultured in Essential
8 Medium by using Corning Synthemax under feeder-free conditions).
The KhES3 cells were induced into pancreatic bud cells according to
the protocol shown in FIG. 1. An about 70% confluent culture of
human ES cell line KhES3 in a culture dish was used. The cells were
detached from the culture dish with CTK solution (ReproCELL Inc.)
and then, dissociated into single cells with Accutase (Innovative
Cell Technologies). Thus obtained single cells were seeded on 24
well- or 6 well-plate (Greiner) coated with Matrigel (BD
Biosciences) at a density of 2.0.times.10.sup.3 cells/well to
3.0.times.10.sup.3 cells/well. Then, the cells were induced to
differentiate into pancreatic bud cells according to the following
procedures.
(Stage 1)
[0148] The cells were exposed to RPMI 1640 Medium (Nacalai Tesque,
Inc.) (0.4 ml/well) supplemented with 2% B-27 (Life Technologies),
100 ng/ml of activin A (R&D systems), 3 .mu.M of CHIR99021
(Axon Medchem) and 10 .mu.M of Y-27632 (Wako Pure Chemical
Industries, Ltd.) for one day. The medium was exchanged with RPMI
1640 Medium (0.8 ml/well) supplemented with 100 ng/ml of activin A
and 2% B-27 (Life Technologies), and the cells were cultured for
two days. The medium was exchanged with RPMI 1640 Medium (0.4
ml/well) supplemented with 100 ng/ml of activin A and 2% B-27 (Life
Technologies), and the cells were cultured for one another day.
(Stage 2)
[0149] The cells were treated with Improved MEM Zinc Option
(Invitrogen) (0.8 ml/well) containing 50 ng/ml of KGF (R&D
systems) and 1% B-27 (Life Technologies) for three days. The medium
was exchanged with Improved MEM Zinc Option (0.8 ml/well)
containing 0-50 ng/ml of KGF, 100 ng/ml of Noggin (Peprotech), 5 nM
or 10 nM of TTNPB (Santa Cruz Biotechnology), 0.5 .mu.M of
3-Keto-N-aminoethyl-N'-aminocaproyldihydrocinnamoyl Cyclopamine
(KAAD-cyclopamine or K-CYC) (Toronto Research Chemicals) and 1%
B-27, and the cells were cultured for three days.
(Stage 3)
[0150] The cells obtained in Stage 2 were dissociated into single
cells with trypsin and seeded on a Low Attachment 96-well plate
(Lipidure Coat, NOF) at a density of 3.0.times.10.sup.3 to
3.0.times.10.sup.4 cells/well. Improved MEM Zinc Option
(15.times.10.sup.4 cells/ml) containing 100 ng/ml of KGF, 100 ng/ml
of Noggin, 50 ng/ml of EGF (R&D systems), 10 .mu.M of Y-27632
and 1% B-27 was added to the plate, and the cells were further
cultured for 4 to 20 days. During this time period, the medium was
exchanged with fresh medium every 4 days. As a control, cells
obtained in Stage 2 were dissociated into single cells with trypsin
and seeded on a Matrigel coated 24 well plate (Greiner) at a
density of 6.0.times.10.sup.4 to 4.8.times.10.sup.3 cells/cm.sup.2
and cultured under adhesive conditions in the same medium for four
days. As another control, cells obtained in Stage 2 were not
dissociated, the medium was exchanged with the same medium as
above, and cultured for four days.
[0151] The cells obtained at four days after the start of Stage 3
(S3d4) were treated with BD Cytofix/Cytoperm.TM. Kit, stained with
an anti-PDX1 antibody (R&D systems) and an anti-NKX6.1 antibody
(University of Iowa), and the ratio of PDX1+ cells as well as the
ratio of PDX1.sup.+/NKX6.1.sup.+ cells were determined by using a
flow cytometer. The ratio of PDX1.sup.+/NKX6.1.sup.+ cells
increased (FIG. 2) in the cell aggregate obtained by culturing
single cells at Stage 3. In addition, it was confirmed that a cell
aggregate was sufficiently constituted by 3.0.times.10.sup.3 or
more cells.
EXAMPLE 2
[0152] Examination using Various iPS Cell Lines
[0153] Four induced pluripotent stem (iPS) cell lines 585A1, 604B1,
692D2 and 648B1 established from peripheral blood mononuclear
cells, (all described in Okita K, et al, Stem Cells. 2013 31:
458-466)) as well as an iPS cell line 409B2 stablished from
fibroblast (Okita K, et al, Nat Methods. 2011 8: 409-412) are
available from Center for iPS Cell Research and Application, Kyoto
University. These iPS cell lines were obtained from Kyoto
University and the cells were induced to differentiate into
pancreatic bud cells according to the same procedures as described
above.
[0154] As a result, a higher ratio of PDX1.sup.+/NKX6.1.sup.+ cells
was obtained in the group where the cells were cultured under a
condition which caused formation of cellular aggregates (Agg) than
where the cells were cultured in adherent cultures (2D) during
Stage 3, in all types of iPS cell lines used as starting materials
(FIG. 3).
EXAMPLE 3
Study on Time Period for Culture
[0155] A human ES cell line KhES3 was used. In Stage 3, cells were
cultured for 0 day (S3d0), 1 day (S3d1), 2 days (S3d2), 4 days
(S3d4), 8 days (S3d8), 12 days (S3d12), 16 days (S3d16) or 20 days
(S3d20). In the cell culture obtained by culturing the cells in
Stage 3 for 8 days or more, the ratio of PDX1.sup.+/NKX6.1.sup.+
cells increased. In this examination, the ratio of
PDX1.sup.+/NKX6.1.sup.+ cells was highest at around day 12 of Stage
3. Further culture of the cells did not significantly decrease the
efficiency (FIGS. 4A and 4B).
[0156] Cellular aggregates were formed from human ES cell line
KhES3. Stage 3 was started with 3.0.times.10.sup.4 cells/well and
the cells were cultured for 4 days (S3d4) or 12 days (53d12). Then,
the cellular aggregates were subjected to immunostaining with
antibodies against markers for pancreatic bud cells (PDX1 , NKX6.1,
SOX9 and GATA4) and those against markers for endocrine cells (INS,
GCG, Somatostatin and Ghrelin) (FIG. 5 and FIG. 6). On day 12 after
starting the formation of cellular aggregates (S3d12), cells
expressing markers for pancreatic bud cells increased and a lot of
cells expressing markers for endocrine cells appeared.
[0157] On day 4 of Stage 3, the cells were cultured in Improved MEM
Zinc Option containing 1% B-27 (15.times.10.sup.4 cells/ml) or in
Improved MEM Zinc Option containing 100 ng/ml of KGF, 50 ng/ml of
EGF (R&D systems) and 1% B-27 (15.times.10.sup.4 cells/ml). In
either case, the ratio of PDX1.sup.+/NKX6.1.sup.+ cells was higher
on day 12 than day 4 of Stage 3 (FIG. 7B). It has been concluded
that the cell destiny is decided by culturing the cells for 4 or
more days in Stage 3. Accordingly, it is desirable that Stage 3 is
performed for at least 4 days.
Study of the effect of ALKS inhibitor II
[0158] The addition of ALKS inhibitor II (Santa Cruz) in Stage 3 on
day 2 or after for 1-2 days increased the ratio of
PDX1.sup.+/NKX6.1.sup.+ cells (FIG. 8B) (FIG. 8A).
EXAMPLE 4
Evaluation of Pancreatic Bud Cells
[0159] Cellular aggregates on day 12 of Stage 3 were collected and
were implanted into the adipose tissues surrounding epididymis of
an immunodeficient mouse (NOD. CB17-Prkdc.sup.scid/J) (Charles
River). Thirty days after the implantation, the implanted tissue
was collected. In the obtained tissue, insulin positive cell
clusters were generated adjacent to PDX1+ cells which formed
tubular structures. It was confirmed that the induced pancreatic
bud cells formed structures reminiscent of pancreatic epithelia in
vivo (FIG. 9).
EXAMPLE 5
Therapeutic Effects of the Induced Pancreatic Bud Cells
[0160] Cellular aggregates on day 4, 5 or 12 of Stage 3 were
collected and washed with physiological saline to prepare
concentrates of cellular aggregates from which the medium was
removed or concentrates of cellular aggregates from which the
culture supernatant was removed.
[0161] Besides these, cellular aggregates on day 4 of Stage 3 were
collected, and added with Improved MEM Zinc Option supplemented
with 1% B-27 and 5 .mu.M of ALKS inhibitor II (Santa Cruz) to give
15.times.10.sup.4 cells/ml suspension and cultured for one day.
Then, cultured cells were washed with physiological saline to
prepare concentrates of cellular aggregates from which the medium
was removed or concentrates of cellular aggregates from which the
culture supernatant was removed.
[0162] The obtained concentrates of cellular aggregates were
implanted into the kidney subcapsule of immunodeficient mice (NOD.
CB17-Prkdc.sup.scid/J).
[0163] The peripheral blood was collected from the implanted mice
at 150 days after the kidney subcapsular implantation of pancreatic
bud cells, and the plasma human C-Peptide levels were measured with
an ELISA kit (Mercodia). Human C-Peptide was confirmed in 13 out of
mice. Each of the concentrated cellular aggregates prepared by
above-described procedures were implanted and at 150 days after the
implantation, peripheral blood human C-Peptide was determined. In
all concentrated cellular aggregates examined, Human C-Peptide was
confirmed in the mouse plasma after 150 days from the implantation
into the kidney. The level of C-Peptide increased with time from 30
days after implantation to 150 days after implantation (FIG.
10A).
[0164] 3 g/kg of glucose was intraperitoneally administered to the
mice after 5 hours or more of fasting at 150 days after
implantation, and the increase in human C-Peptide was measured. The
plasma C-Peptide level increased significantly in 5 out of 9
glucose challenged mice (FIG. 10B).
[0165] The above results demonstrated that the induced pancreatic
bud cells administered to mice engrafted in vivo and thereafter
functioned to produce insulin in response to the blood glucose
level. Accordingly, it was suggested that the induced pancreatic
bud cells can be used as a therapeutic agent for insulin
hyposecretion.
EXAMPLE 6
Study on Protocol in Adherent Cultures (FIG. 11A)
(Stage 1)
[0166] Cells of iPS cell line 585A1 were seeded on a Matrigel
coated 24 well plate at a density of 2.0.times.10.sup.5 cells/well,
and cultured in RPMI 1640 Medium (Nacalai Tesque, Inc.)
supplemented with 2% B-27 (Life Technologies), 100 ng/ml of activin
A (R&D systems), 3 .mu.M of CHIR99021 (Axon Medchem) and 10
.mu.M of Y-27632 (Wako Pure Chemical Industries, Ltd.) for one day.
The medium was exchanged with RPMI 1640 Medium containing 100 ng/ml
of activin A, 1 .mu.M of CHIR99021 and 2% B-27 (Life Technologies),
and the cells were cultured for two days. The medium was then
exchanged with RPMI 1640 Medium containing 100 ng/ml of activin A
and 2% B-27 (Life Technologies), and the cells were cultured for
one day.
(Stage 2)
[0167] The medium of the culture obtained in Stage 1 was exchanged
with Improved MEM Zinc Option (Invitrogen) containing 50 ng/ml of
KGF (R&D systems) and 1% B-27 (Life Technologies) and the cells
were cultured for three days. Subsequently, the medium was
exchanged with Improved MEM Zinc Option containing 50 ng/ml of KGF,
100 ng/ml of Noggin (Peprotech), 10 nM of TTNPB (Santa Cruz
Biotechnology), 0.5 .mu.M of
3-Keto-N-aminoethyl-N'-aminocaproyldihydrocinnamoyl Cyclopamine
(KAAD-cyclopamine or K-CYC) (Toronto Research Chemicals) and 1%
B-27, and the cells were cultured for three days.
(Stage 3)
[0168] The cells obtained in Stage 2 were dissociated into single
cells with trypsin, and seeded on a Matrigel coated 24 well plate
at a density of 1.6.times.10.sup.5 to 2.4.times.10.sup.3
cells/cm.sup.2. Improved MEM Zinc Option containing 100 ng/ml of
KGF, 100 ng/ml of Noggin, 50 ng/ml of EGF (R&D systems),
various concentration of Y-27632 and 1% B-27 (15.times.10.sup.4
cells/ml) was added to the plate and the cells were cultured for
four days.
[0169] The obtained cells were treated with BD Cytofix/Cytoperm.TM.
Kit, and thereafter, the ratio of PDX1.sup.+/NKX6.1.sup.+ cells was
detected by staining the cells with an anti-PDX1 antibody (R&D
systems) and an anti-NKX6.1 antibody (University of Iowa) and
analyzing by flow cytometer.
[0170] When Y-27632 was used in Stage 3, the ratio of
PDX1.sup.+/NKX6.1.sup.+ cells increased in a
concentration-dependent manner, and this effect was highest at 100
.mu.M of Y-27632 (FIGS. 11B and C).
[0171] As described above, PDX1.sup.+NKX6.1.sup.+ cells were
produced in adherent culture in the presence of a ROCK inhibitor in
Stage 3, after the cells were dissociated.
EXAMPLE 7
Study on Protocol for Adherent Culture (FIG. 12A) and Additives
During Stage 3
(Stage 1)
[0172] Cells of iPS cell line 585A1 were seeded on a Matrigel
coated 24 well plate at a density of 2.0.times.10.sup.5 cells/well,
and the cells were cultured in RPMI 1640 Medium (Nacalai Tesque,
Inc.) supplemented with 2% B-27 (Life Technologies), to which 100
ng/ml of activin A (R&D systems), 3 .mu.M of CHIR99021 (Axon
Medchem) and 10 .mu.M of Y-27632 (Wako Pure Chemical Industries,
Ltd.) were added, for one day. The medium was exchanged with RPMI
1640 Medium containing 100 ng/ml of activin A, 1 .mu.M of CHIR99021
and 2% B-27 (Life Technologies), and the cells were cultured for
two days. In addition, the medium was exchanged with RPMI 1640
Medium containing 100 ng/ml of activin A and 2% B-27 (Life
Technologies), and the cells were cultured for one day.
(Stage 2)
[0173] The medium was exchanged with Improved MEM Zinc Option
(Invitrogen) containing 50 ng/ml of KGF (R&D systems) and 1%
B-27 (Life Technologies), and the cells were cultured for 3 or 4
days. Subsequently, the medium was exchanged with Improved MEM Zinc
Option containing 50 ng/ml of KGF, 100 ng/ml of Noggin (Peprotech),
10 nM of TTNPB (Santa Cruz Biotechnology), 0.5 .mu.M of
3-Keto-N-aminoethyl-N'-aminocaproyldihydrocinnamoyl Cyclopamine
(KAAD-cyclopamine or K-CYC) (Toronto Research Chemicals) and 1%
B-27 and the cells were cultured for 2 or 3 days.
(Stage 3)
[0174] The cells obtained in Stage 2 were dissociated into single
cells with trypsin, and the cells were seeded on a Matrigel coated
24 well plate at a density of 1.6.times.10.sup.5 to
2.4.times.10.sup.5 cells/cm.sup.2. Improved MEM Zinc Option
containing 50 ng/ml of KGF, 100 ng/ml of Noggin, 10 nM of TTNPB,
0.5 .mu.M of KAAD-cyclopamine, 10 .mu.M of Y-27632 and 1% B-27 was
added to the plate, and the cells were cultured for one day.
Subsequently, the medium was exchanged with Improved MEM Zinc
Option containing 100 ng/ml of KGF, 100 ng/ml of Noggin, 50 ng/ml
of EGF (R&D systems), each of compounds (Y-27632, Fasudil
(HA-1077), SR3677, GSK269962, H-1152 and Blebbistatin) and 1% B-27
(15.times.10.sup.4 cells/ml) and the cells were cultured for
additional 4 days.
[0175] The obtained cells were treated with BD Cytofix/Cytoperm.TM.
Kit, stained with an anti-PDX1 antibody (R&D systems) and an
anti-NKX6.1 antibody (University of Iowa), and the ratio of
PDX1.sup.+ cells as well as the ratio of PDX1.sup.+/NKX6.1.sup.+
cells were determined by using a flow cytometer or an image
analyzer.
[0176] When Fasudil was used in Stage 3, the ratio of
PDX1.sup.+/NKX6.1.sup.+ cells increased in a
concentration-dependent manner, and this effect was highest at 50
.mu.M of Fasudil (FIGS. 12B and C).
[0177] When SR3677 was used in Stage 3, the ratio of NKX6.1+ cells
increased in a concentration-dependent manner, and this effect was
highest at 5 .mu.M of SR3677 (FIGS. 13A and B).
[0178] When GSK269962 was used in Stage 3, the ratio of
NKX6.1.sup.+ cells increased in a concentration-dependent manner,
and this effect was highest at 1 .mu.M of GSK269962 (FIGS. 13C and
D).
[0179] When H-1152 was used in Stage 3, the ratio of NKX6.1.sup.+
cells increased in a concentration-dependent manner, and this
effect was highest at 50 .mu.M of H-1152 (FIGS. 14A and B).
[0180] When Blebbistatin was used in Stage 3, the ratio of
PDX1.sup.+/NKX6.1.sup.+ cells increased from 5 .mu.M of
Blebbistatin, and this effect was high up to 20 .mu.M of
Blebbistatin (FIGS. 14C and D).
[0181] As described above, it was confirmed that the cell ratio of
PDX1.sup.+/NKX6.1.sup.+ cells increased by decomposing cells,
culturing the cells in Stage 3 in adherent cultures by using a
similar medium to that used in Stage 2, and adding a ROCK inhibitor
or a nonmuscle myosin II inhibitor further in Stage 3.
* * * * *