U.S. patent application number 17/653185 was filed with the patent office on 2022-06-16 for method for inducing differentiation of alveolar epithelial cells.
This patent application is currently assigned to KYOTO UNIVERSITY. The applicant listed for this patent is KYOTO UNIVERSITY. Invention is credited to Shimpei GOTOH, Satoshi KONISHI, Michiaki MISHlMA, Yuki YAMAMOTO.
Application Number | 20220186189 17/653185 |
Document ID | / |
Family ID | |
Filed Date | 2022-06-16 |
United States Patent
Application |
20220186189 |
Kind Code |
A1 |
GOTOH; Shimpei ; et
al. |
June 16, 2022 |
METHOD FOR INDUCING DIFFERENTIATION OF ALVEOLAR EPITHELIAL
CELLS
Abstract
This invention provides a method for stably producing type II
alveolar epithelial cells from pluripotent stem cells.
Specifically, the invention relates to a method for producing type
II alveolar epithelial cells from pluripotent stem cells comprising
steps of: (1) culturing pluripotent stem cells in a medium
containing activin A and a GSK3.beta. inhibitor; (2) culturing the
cells obtained in Step (1) in a medium containing a BMP inhibitor
and a TGF.beta. inhibitor; (3) culturing the cells obtained in Step
(2) in a medium containing BMP4, retinoic acid, and a GSK3.beta.
inhibitor; (4) culturing the ventral anterior foregut cells
obtained in Step (3) in a medium containing a GSK3.beta. inhibitor,
FGF10, KGF, and a NOTCH signal inhibitor; and (5) subjecting the
alveolar epithelial progenitor cells obtained in Step (4) to
three-dimensional culture in a medium containing a steroid drug, a
cAMP derivative, a phosphodiesterase inhibitor, and KGF.
Inventors: |
GOTOH; Shimpei; (Kyoto,
JP) ; YAMAMOTO; Yuki; (Kyoto, JP) ; KONISHI;
Satoshi; (Kyoto, JP) ; MISHlMA; Michiaki;
(Kyoto, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KYOTO UNIVERSITY |
Kyoto |
|
JP |
|
|
Assignee: |
KYOTO UNIVERSITY
Kyoto
JP
|
Appl. No.: |
17/653185 |
Filed: |
March 2, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15555183 |
Sep 1, 2017 |
11299712 |
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PCT/JP2016/057254 |
Mar 2, 2016 |
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17653185 |
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International
Class: |
C12N 5/071 20060101
C12N005/071 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 6, 2015 |
JP |
2015-045298 |
Claims
1. A method for type II alveolar epithelial cell culture comprising
a step of subjecting type II alveolar epithelial cells to
three-dimensional culture in a medium containing a steroid drug, a
cAMP derivative, a phosphodiesterase inhibitor, and keratinocyte
growth factor (KGF).
2. The method according to claim 1, wherein the steroid drug is
dexamethasone, the cAMP derivative is 8-Br-cAMP, and the
phosphodiesterase inhibitor is 3-isobutyl-1-methylxanthine
(IBMX).
3. The method according to claim 1, which comprises subjecting type
II alveolar epithelial cells to three-dimensional culture in a
medium further supplemented with a Rho kinase (ROCK) inhibitor.
4. The method according to claim 3, wherein the ROCK inhibitor is
Y-27632.
5. The method according to claim 1, which comprises subjecting type
II alveolar epithelial cells to three-dimensional culture in a
medium further supplemented with a WNT signal inhibitor and/or
insulin like growth factor 2 (IGF2).
6. The method according to claim 5, wherein the WNT signal
inhibitor is WNT inhibitory factor 1 (WIF1).
7. The method according to claim 1, wherein the type II alveolar
epithelial cells are produced by a method for producing a cell
population comprising type II alveolar epithelial cells from
pluripotent stem cells comprising Steps (1) to (5) below: (1)
culturing pluripotent stem cells in a medium containing activin A
and a glycogen synthase kinase 3.beta. (GSK3.beta.) inhibitor; (2)
culturing the cells obtained in Step (1) in a medium containing a
morphogenetic protein (BMP) inhibitor and a transforming growth
factor beta (TGF.beta.) inhibitor; (3) culturing the cells obtained
in Step (2) in a medium containing bone morphogenetic protein 4
(BMP4), retinoic acid, and a GSK3.beta. inhibitor; (4) culturing
the ventral anterior foregut cells obtained in Step (3) in a medium
containing a GSK3.beta. inhibitor, Fibroblast Growth Factor 10
(FGF10), keratinocyte growth factor (KGF), and a NOTCH signal
inhibitor for a duration of time until alveolar epithelial
progenitor cells are induced; followed by a step of isolating
carboxypeptidase M-positive (CPM-positive) cells as alveolar
epithelial progenitor cells; and (5) subjecting the induced
alveolar epithelial progenitor cells obtained in Step (4) to
three-dimensional culture in a basal medium supplemented with
additives consisting of a steroid drug, a cAMP derivative, a
phosphodiesterase inhibitor, and KGF; following Step (5), a further
step of isolating cells positive for one or more type II alveolar
epithelial cell markers selected from the group consisting of
surfactant protein C (SFTPC), epithelial cell adhesion molecule
(EpCAM), and carcinoembryonic antigen-related cell adhesion
molecule 6 (CEACAM6) as type II alveolar epithelial cells, wherein
cells positive for staining of acidic fractions for live cells are
isolated as type II alveolar epithelial cells; thereby obtaining a
cell population, wherein the type II alveolar epithelial cells are
at least 50% of the cell population, relative to total epithelial
cells.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 15/555,183, filed Sep. 1, 2017 which in turn is a 371 of
PCT/JP2016/057254, filed Mar. 2, 2016, which claims the benefit of
Japanese Patent Application No. 2015-045298, filed Mar. 6, 2015,
the contents of each of which are incorporated herein by
reference.
TECHNICAL FIELD
[0002] The present invention relates to a method for producing type
II alveolar epithelial cells from pluripotent stem cells, a kit for
producing type II alveolar epithelial cells from pluripotent stem
cells, a method for type II alveolar epithelial cell culture, and a
kit for type II alveolar epithelial cell culture, for example.
BACKGROUND ART
[0003] The lung is one of the most complicated organs, and it is
considered to be composed of approximately 40 different types of
cells. Among them, the pulmonary alveolus is composed of the
alveolar space, which stores gas, and the alveolar epithelium,
which surrounds the same. In addition, the alveolar epithelium is
composed of the type I alveolar epithelial cells and the type II
alveolar epithelial cells. The former forms a blood-air barrier
with the microvascular endothelium surrounding the pulmonary
alveolus with the aid of the basal membrane and exchanges the
intra-alveolar gas with the blood gas. The latter comprises many
lamellar corpuscles, it undergoes exocytosis of pulmonary
surfactants, and it forms the alveolar lining layer.
[0004] In recent years, cells having pluripotency, such as
embryonic stem cells (ES cells) or induced pluripotent stem cells
(iPS cells) obtained by introducing undifferentiated-cell-specific
genes into somatic cells, have been reported, methods for inducing
alveolar epithelial cells from such cells have been reported
(Rippon, H. J. et al, Cloning Stem Cells 6: 49-56, 2004; Coraux, C.
et al, Am. J. Respir. Cell Mol. Biol., 32: 87-92, 2005; Morrisey,
E. E. and Hogan, B. L. M., Dev. Cell., 18: 8-23, 2010; Ghaedi, M.
et al., J. Clin. Invest., Vol. 123, pp. 4950-62, 2013; and Huang,
S. X. et al., Nat. Biotechnol., Vol. 32, pp. 84-91, 2014), and
growth factors and the like that are necessary for the induction of
such cells have also been reported. However, there are no examples
demonstrating the induction of human pulmonary alveolar cells with
high reproducibility and efficiency.
[0005] The present inventors disclose that three-dimensional
coculture of human pluripotent stem cells is useful for induction
of differentiation into type II alveolar epithelial cells and a
reporter enables isolation of type II alveolar epithelial cells
(Gotoh, S. et al., Stem Cell Reports, 2014, Vol. 3, pp. 394-403).
The present inventors also disclose a method for producing alveolar
epithelial progenitor cells from pluripotent stem cells (WO
2014/168264).
SUMMARY OF THE INVENTION
[0006] It is an object of the present invention to provide a method
for producing type II alveolar epithelial cells from pluripotent
stem cells and a kit for producing type II alveolar epithelial
cells from pluripotent stem cells. It is another object of the
present invention to provide a method for type II alveolar
epithelial cell culture and a kit for type II alveolar epithelial
cell culture.
[0007] The present inventors have conducted concentrated studies in
order to attain the above objects. As a result, they discovered
that pluripotent stem cells could be induced to differentiate into
type II alveolar epithelial cells with the use of various growth
factors and compounds. This has led to the completion of the
present invention.
[0008] Specifically, the present invention includes the following.
[0009] [1] A method for producing type II alveolar epithelial cells
from pluripotent stem cells comprising Steps (1) to (5) below:
[0010] (1) culturing pluripotent stem cells in a medium containing
activin A and a GSK3.beta. inhibitor;
[0011] (2) culturing the cells obtained in Step (1) in a medium
containing a BMP inhibitor and a TGF.beta. inhibitor;
[0012] (3) culturing the cells obtained in Step (2) in a medium
containing BMP4, retinoic acid, and a GSK3.beta. inhibitor;
[0013] (4) culturing the ventral anterior foregut cells obtained in
Step (3) in a medium containing a GSK3.beta. inhibitor, FGF10, KGF,
and a NOTCH signal inhibitor; and
[0014] (5) subjecting the alveolar epithelial progenitor cells
obtained in Step (4) to three-dimensional culture in a medium
containing a steroid drug, a cAMP derivative, a phosphodiesterase
inhibitor, and KGF. [0015] [2] The method according to [1], wherein
the GSK3.beta. inhibitor is CHIR99021, the BMP inhibitor is Noggin,
the TGF.beta. inhibitor is SB431542, the NOTCH signal inhibitor is
DAPT, the steroid drug is dexamethasone, the cAMP derivative is
8-Br-cAMP, and the phosphodiesterase inhibitor is
3-isobutyl-1-methylxanthine (IBMX). [0016] [3] The method according
to [1] or [2], wherein Step (1) comprises culturing pluripotent
stem cells in a medium further supplemented with a ROCK inhibitor
and/or a HDAC inhibitor. [0017] [4] The method according to [3],
wherein the ROCK inhibitor is Y-27632 and/or the HDAC inhibitor is
sodium butyrate. [0018] [5] The method according to any one of [1]
to [4], which further comprises, following Step (4), a step of
isolating CPM-positive cells as alveolar epithelial progenitor
cells. [0019] [6] The method according to any one of [1] to [5],
wherein, following Step (4), the alveolar epithelial progenitor
cells are cryopreserved and the alveolar epithelial progenitor
cells cultured in Step (5) are obtained by thawing the
cryopreserved alveolar epithelial progenitor cells. [0020] [7] The
method according to any one of [1] to [6], wherein Step (5)
comprises subjecting alveolar epithelial progenitor cells to
three-dimensional culture in a medium further supplemented with a
ROCK inhibitor. [0021] [8] The method according to [7], wherein the
ROCK inhibitor is Y-27632. [0022] [9] The method according to any
one of [1] to [8], which further comprises, following Step (5), a
step of isolating cells positive for one or more type II alveolar
epithelial cell markers selected from the group consisting of
SFTPC, EpCAM, and CEACAM6 as type II alveolar epithelial cells.
[0023] [10] The method according to [9], wherein the cells further
positive for staining of acidic fractions for live cells are
isolated as type II alveolar epithelial cells. [0024] [11] The
method according to any one of [1] to [10], wherein, following Step
(5), the type II alveolar epithelial cells obtained are
cryopreserved. [0025] [12] A kit for producing type II alveolar
epithelial cells from pluripotent stem cells comprising activin A,
a GSK3.beta. inhibitor, a BMP inhibitor, a TGF.beta. inhibitor,
BMP4, retinoic acid, FGF10, KGF, a NOTCH signal inhibitor, a
steroid drug, a cAMP derivative, and a phosphodiesterase inhibitor.
[0026] [13] The kit according to [12], wherein the GSK3.beta.
inhibitor is CHIR99021, the BMP inhibitor is Noggin, the TGF.beta.
inhibitor is SB431542, the NOTCH signal inhibitor is DAPT, the
steroid drug is dexamethasone, the cAMP derivative is 8-Br-cAMP,
and the phosphodiesterase inhibitor is IBMX. [0027] [14] The kit
according to [12] or [13], which further comprises a ROCK inhibitor
and/or a HDAC inhibitor. [0028] [15] The kit according to [14],
wherein the ROCK inhibitor is Y-27632 and/or the HDAC inhibitor is
sodium butyrate. [0029] [16] A method for type II alveolar
epithelial cell culture comprising a step of subjecting type II
alveolar epithelial cells to three-dimensional culture in a medium
containing a steroid drug, a cAMP derivative, a phosphodiesterase
inhibitor, and KGF. [0030] [17] The method according to [16],
wherein the steroid drug is dexamethasone, the cAMP derivative is
8-Br-cAMP, and the phosphodiesterase inhibitor is IBMX. [0031] [18]
The method according to [16] or [17], which comprises subjecting
type II alveolar epithelial cells to three-dimensional culture in a
medium further supplemented with a ROCK inhibitor. [0032] [19] The
method according to [18], wherein the ROCK inhibitor is Y-27632.
[0033] [20] The method according to any one of [16] to [19], which
comprises subjecting type II alveolar epithelial cells to
three-dimensional culture in a medium further supplemented with a
WNT signal inhibitor and/or IGF2. [0034] [21] The method according
to [20], wherein the WNT signal inhibitor is WIF1. [0035] [22] The
method according to any one of [16] to [21], wherein the type II
alveolar epithelial cells are produced by the method according to
any one of [1] to [11]. [0036] [23] A kit for type II alveolar
epithelial cell culture comprising a steroid drug, a cAMP
derivative, a phosphodiesterase inhibitor, and KGF. [0037] [24] The
kit according to [23], wherein the steroid drug is dexamethasone,
the cAMP derivative is 8-Br-cAMP, and the phosphodiesterase
inhibitor is IBMX. [0038] [25] The kit according to [23] or [24],
which further comprises a ROCK inhibitor. [0039] [26] The kit
according to [25], wherein the ROCK inhibitor is Y-27632. [0040]
[27] The kit according to any one of [23] to [26], which further
comprises the WNT signal inhibitor and/or IGF2. [0041] [28] The kit
according to [27], wherein the WNT signal inhibitor is WIF1.
[0042] This description includes part or all of the content as
disclosed in Japanese Patent Application No. 2015-045298, which is
a priority document of the present application.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0044] FIG. 1 shows a method for inducing type II alveolar
epithelial cells from ventral anterior foregut cells using human
pluripotent stem cells.
[0045] FIGS. 2A and 2B show the results of examination of the
composition of the medium for Step 4 shown in FIG. 1 in terms of
the efficiency for alveolar epithelial progenitor cell induction
and isolation using NKX2.1 as a marker protein.
[0046] FIGS. 2C and 2D are continued from FIGS. 2A and 2B.
[0047] FIG. 3 shows the results of examination of the composition
of the medium for Step 4 in terms of the expression level of SFTPC,
which is a marker protein for type II alveolar epithelial cells, on
Day 35 (i.e., upon completion of Step 5).
[0048] FIG. 4 shows the results of examination of the culture
period under the conditions shown in FIG. 2 (4) in terms of the
expression level of SFTPC, which is a marker protein for type II
alveolar epithelial cells, on Day 35 (i.e., upon completion of Step
5).
[0049] FIG. 5 shows the results of whole well imaging demonstrating
a process comprising isolating CPM.sup.+ cells (alveolar epithelial
progenitor cells) separately from the control CPM.sup.- cells using
the SFTPC (SFTPC-GFP) reporter iPS cells on Day 21 (i.e., upon
completion of Step 4), subjecting the isolated cells to
three-dimensional coculture with human fetal lung fibroblasts, and
inducing type II alveolar epithelial cells from alveolar epithelial
progenitor cells over a period of 14 days.
[0050] FIG. 6 shows photographs demonstrating the results of
observation of spheroids formed from CPM.sup.+ cells on Day 35
(i.e., upon completion of Step 5) in a high-power field.
[0051] FIG. 7 shows the results of flow cytometry conducted on Day
35 (i.e., upon completion of Step 5) demonstrating that the
proportion of SFTPC-GFP.sup.+ cells (i.e., type II alveolar
epithelial cells) reached 50% of the CPM.sup.+ cell-derived
EpCAM.sup.+ cells.
[0052] FIG. 8 demonstrates that both the SFTPC and SFTPB expression
levels were increased by the method for type II alveolar epithelial
cell induction shown in FIG. 1 (the new protocol), compared with
the method described in Gotoh, S. et al., Stem Cell Reports, 2014,
Vol. 3, pp. 394-403 (the previously published protocol).
[0053] FIG. 9 demonstrates that various marker proteins for type II
alveolar epithelial cells are expressed in a CPM.sup.+ cell-derived
three-dimensional culture product on Day 35 (i.e., upon completion
of Step 5), in comparison with the case of a CPM.sup.- cell-derived
three-dimensional culture product.
[0054] FIG. 10 shows double fluorescent immunostaining images of
spheroids on Day 35 (i.e., upon completion of Step 5).
[0055] FIG. 11 shows transmission electron microscopic images of
spheroids on Day 35 (i.e., upon completion of Step 5).
[0056] FIG. 12 shows a method for culturing the type II alveolar
epithelial cells induced from human pluripotent stem cells via
subculture in a three-dimensional coculture system over a long
period of time.
[0057] FIG. 13 demonstrates that SFTPC could be maintained at a
certain positive rate via subculture of type II alveolar epithelial
cells in a three-dimensional coculture system.
[0058] FIG. 14 shows double fluorescent immunostaining images
demonstrating that the spheroids formed by the type II alveolar
epithelial cells subjected to subculture (AT2-P3) also expressed
DC-LAMP and SFTPC as with the case of spheroids at the AT2-P0
stage.
[0059] FIG. 15 demonstrates that type II alveolar epithelial cells
were isolated via induction of differentiation with the use of the
human iPS cell line (604A1) instead of SFTPC-GFP reporter cells up
to Day 35 (i.e., upon completion of Step 5) and with the use of the
anti-EpCAM antibody and LysoTracker.RTM. at the AT2-P0 stage.
[0060] FIG. 16 demonstrates that type II alveolar epithelial cells
were isolated via induction of differentiation with the use of the
human iPS cell line (604A1) instead of SFTPC-GFP reporter cells up
to Day 35 (i.e., upon completion of Step 5) as with the case shown
in FIG. 15 and with the use of the anti-CEACAM6 antibody at the
AT2-P0 stage.
[0061] FIG. 17 demonstrates that type II alveolar epithelial cells
were efficiently maintained in the medium for Step 5 supplemented
with WIF1 (300 ng/ml) or IGF2 (100 ng/ml), when isolating AT2-P0
with the use of the SFTPC-GFP reporter cells and subjecting the
isolated cells to subculture as shown in FIG. 12.
[0062] FIG. 18 shows a method for inducing type II alveolar
epithelial cells by cryopreserving human alveolar epithelial
progenitor cells on Day 21 (i.e., upon completion of Step 4) and
subjecting the resulting cells to three-dimensional coculture.
[0063] FIG. 19 demonstrates that type II alveolar epithelial cells
were differentiated from the cryopreserved alveolar epithelial
progenitor cells with an efficiency of approximately 20% as a
result of the process shown in FIG. 18.
[0064] FIG. 20 shows a method for type II alveolar epithelial cell
culture comprising cryopreserving type II alveolar epithelial cells
and subjecting the resulting cells to three-dimensional
coculture.
EMBODIMENTS FOR CARRYING OUT THE INVENTION
[0065] [Method for Producing Type II Alveolar Epithelial Cells from
Pluripotent Stem Cells]
[0066] The method for producing type II alveolar epithelial cells
from pluripotent stem cells according to the present invention
comprises Steps (1) to (5) below:
[0067] (1) culturing pluripotent stem cells in a medium containing
activin A and a GSK3.beta. inhibitor;
[0068] (2) culturing the cells obtained in Step (1) in a medium
containing a BMP inhibitor and a TGF.beta. inhibitor;
[0069] (3) culturing the cells obtained in Step (2) in a medium
containing BMP4, retinoic acid, and a GSK3.beta. inhibitor;
[0070] (4) culturing the ventral anterior foregut cells obtained in
Step (3) in a medium containing a GSK3.beta. inhibitor, FGF10, KGF,
and a NOTCH signal inhibitor; and
[0071] (5) subjecting the alveolar epithelial progenitor cells
obtained in Step (4) to three-dimensional culture in a medium
containing a steroid drug, a cAMP derivative, a phosphodiesterase
inhibitor, and KGF.
[0072] In the present invention, the term "ventral anterior foregut
cells" refers to cells that are destined to differentiate into the
thyroid gland or lung in the presence of developmentally
appropriate stimuli, and such cells express NKX2-1, GATA6, and/or
HOPX.
[0073] In the present invention, the term "alveolar epithelial
progenitor cells" refers to progenitor cells of type I alveolar
epithelial cells or type II alveolar epithelial cells, which
express CPM and/or NKX2-1.
[0074] In the present invention, the term "type II alveolar
epithelial cells" refers to epithelial cells that histologically
produce pulmonary surfactants and have morphological features, such
as lamellae and multivesicular bodies, in the cells. For example,
SFTPA, SFTPB, SFTPC, SFTPD, EpCAM, CEACAM6, DC-LAMP, ABCA3, and
LPCAT1 are expressed therein.
[0075] The steps of the method for producing type II alveolar
epithelial cells from pluripotent stem cells according to the
present invention are described below.
(1) Step of Culture in a Medium Containing Activin A and a
GSK3.beta. Inhibitor (Step 1)
[0076] A medium used in the step of pluripotent stem cell culture
according to the present invention can be prepared from a medium
used for animal cell culture as a basal medium. Examples of basal
media include IMDM medium, Medium 199, Eagle's Minimum Essential
Medium (EMEM), .alpha.MEM medium, Dulbecco's modified Eagle's
Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's
medium, NEUROBASAL.RTM. Medium (Life Technologies), and a mixture
of any such media. A medium may or may not contain blood serum. A
medium may optionally contain one or more serum substitutes
selected from among, for example, albumin, transferrin, Knockout
Serum Replacement (KSR) (an FBS serum substitute used for ES cell
culture), N2 supplements (Invitrogen), B27 supplements
(Invitrogen), fatty acid, insulin, collagen precursors, trace
elements, 2-mercaptoethanol, and 3'-thiol glycerol. In addition, a
medium can contain one or more substances selected from among, for
example, lipids, amino acids, L-glutamine, GLUTAMAX.TM.
(Invitrogen), nonessential amino acids, vitamins, growth factors,
low-molecular-weight compounds, antibiotics, antioxidants, pyruvic
acids, buffer agents, and inorganic salts. RPMI 1640 medium
supplemented with B27 and antibiotics is preferable.
[0077] In this step, pluripotent stem cells are cultured in a
medium prepared by supplementing the basal medium described above
with activin A and a GSK3.beta. inhibitor. In this step, a HDAC
inhibitor may further be added.
[0078] Activin A is a homodimer with two beta A chains, the amino
acid sequence of activin A is 100% homologous to that of a protein
of a human, mouse, rat, pig, cow, or cat, and, accordingly,
relevant species are not particularly limited. In the present
invention, activin A is preferably of an active form with the
N-terminal peptide being cleaved, and it is preferably a homodimer
comprising, bound thereto via a disulfide bond, the Gly311-Ser426
fragment with the N-terminal peptide of the inhibin beta A chain
(e.g., NCBI Accession Number NP 002183) being cleaved. Such activin
A is commercially available from, for example, Wako and R&D
Systems.
[0079] The activin A concentration in a medium is, for example, 10
ng/ml to 1 mg/ml, and it is specifically 10 ng/ml, 20 ng/ml, 30
ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml,
100 ng/ml, 150 ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml,
600 ng/ml, 700 ng/ml, 800 ng/ml, 900 ng/ml, or 1 mg/ml, although
the concentration is not limited thereto. The concentration is
preferably 100 ng/ml.
[0080] The term "GSK3.beta. inhibitor" used herein is defined as a
substance that inhibits kinase activity of the GSK-3.beta. protein
(e.g., the capacity for phosphorylation of .beta.-catenin), and
many such substances are already known. Examples thereof include:
an indirubin derivative, such as BIO, which is also known as a
GSK-3.beta. inhibitor IX (6-bromoindirubin-3'-oxime); a maleimide
derivative, such as SB216763
(3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione);
a phenyl .alpha.-bromomethylketone compound, such as a GSK-3.beta.
inhibitor VII (4-dibromoacetophenone); a cell-permeable
phosphorylated peptide, such as L803-mts, which is also known as a
GSK-3.beta. peptide inhibitor (i.e., Myr-N-GKEAPPAPPQSpP-NH.sub.2);
and CHIR99021, such as
6-[2-[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-yla-
mino]-ethylamino]pyridine-3-carbonitrile, with high selectivity.
While such compounds are commercially available from, for example,
Calbiochem or Biomol, and easily used, such compounds may be
obtained from other companies, or persons may prepare such
compounds by themselves.
[0081] A GSK-3.beta. inhibitor that can be preferably used in the
present invention is CHIR99021. In this step, the CHIR99021
concentration in a medium is, for example, 1 nM to 50 .mu.M, and it
is specifically 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 750 nM, 1
.mu.M, 1.5 .mu.M, 2 .mu.M, 2.5 .mu.M, 3 .mu.M, 3.5 .mu.M, 4 .mu.M,
4.5 .mu.M, 5 .mu.M, 6 .mu.M, 7 .mu.M, 8 .mu.M, 9 .mu.M, 10 .mu.M,
15 .mu.M, 20 .mu.M, 25 .mu.M, 30 .mu.M, 40 .mu.M, or 50 .mu.M,
although the concentration is not limited thereto. In this step,
the concentration is preferably 1 .mu.M.
[0082] The term "HDAC inhibitor" is defined as a substance that
inhibits or inactivates enzyme activity of histone deacetylase
(HDAC). Examples thereof include: low-molecular-weight inhibitors,
such as valproic acid (VPA) (Nat. Biotechnol., 26 (7): 795-797,
2008), trichostatin A, sodium butyrate (NaB), MC 1293, and M344;
nucleic acid-based expression inhibitors, such as siRNAs and shRNAs
against HDAC (e.g., HDAC1 siRNA Smartpool.RTM. (Millipore) and HuSH
29 mer shRNA Constructs against HDAC1 (OriGene)); and DNA
methyltransferase inhibitors (e.g., 5'-azacytidine) (Nat.
Biotechnol., 26 (7): 795-797, 2008).
[0083] An HDAC inhibitor that can be preferably used in the present
invention is sodium butyrate (NaB). The sodium butyrate (NaB)
concentration in a medium is, for example, 1 .mu.M to 5 mM, and it
is specifically 1 .mu.M, 10 .mu.M, 50 .mu.M, 100 .mu.M, 125 .mu.M,
250 .mu.M, 500 .mu.M, 750 .mu.M, 1 mM, 2 mM, 3 mM, 4 mM, or 5 mM,
although the concentration is not limited thereto. The
concentration is preferably 125 .mu.M to 250 .mu.M.
[0084] In this step, culture may be conducted in a culture vessel
treated with a coating agent. A coating agent may be a naturally
occurring or artificially synthesized extracellular matrix.
Examples thereof include BD MATRIGEL.RTM., collagen, gelatin,
laminin, heparan sulfate proteoglycan, entactin, and a combination
of any thereof, with MATRIGEL.RTM. being preferable.
[0085] This step may comprise a process of pluripotent stem cell
detachment. Examples of methods for cell detachment include a
method of mechanical detachment and a method of cell detachment
involving the use of a cell detachment solution having protease
activity and collagenase activity (e.g., Accutase.TM. and
Accumax.TM.) or a cell detachment solution having collagenase
activity alone. It is preferable that human pluripotent stem cells
be detached with the use of a cell detachment solution having
protease activity and collagenase activity, with the use of
Accutase.TM. being particularly preferable.
[0086] When the step comprises a process of cell detachment, a ROCK
inhibitor may be added to a medium, so as to inhibit pluripotent
stem cell death caused by detachment.
[0087] An ROCK inhibitor is not particularly limited, provided that
it can inhibit functions of Rho kinase (ROCK). Examples thereof
include: Y-27632
((+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide
dihydrochloride) (e.g., Ishizaki et al., Mol. Pharmacol., 57,
976-983, 2000; Narumiya et al., Methods Enzymol., 325, 273-284,
2000); Fasudil/HA1077 (e.g., Uenata et al., Nature 389: 990-994,
1997); H-1152 (e.g., Sasaki et al., Pharmacol. Ther., 93: 225-232,
2002); Wf-536 (e.g., Nakajima et al., Cancer Chemother. Pharmacol.,
52 (4): 319-324, 2003) and derivatives thereof; antisense nucleic
acids against ROCK; RNA interference-inducible nucleic acids (e.g.,
siRNA); dominant-negative variants; and expression vectors thereof.
Since other low-molecular-weight compounds are known as ROCK
inhibitors, such compounds and derivatives thereof can also be used
in the present invention (e.g., U.S. Patent Application Publication
Nos. 2005/0209261, 2005/0192304, 2004/0014755, 2004/0002508,
2004/0002507, 2003/0125344, and 2003/0087919, WO 2003/062227, WO
2003/059913, WO 2003/062225, WO 2002/076976, and WO 2004/039796).
In the present invention, one or more types of ROCK inhibitors can
be used.
[0088] An ROCK inhibitor that can be preferably used in the present
invention is Y-27632. The Y-27632 concentration is, for example,
100 nM to 50 .mu.M, and it is specifically 100 nM, 500 nM, 750 nM,
1 .mu.M, 2 .mu.M, 3 .mu.M, 4 .mu.M, 5 .mu.M, 6 .mu.M, 7 .mu.M, 8
.mu.M, 9 .mu.M, 10 .mu.M, 15 .mu.M, 20 .mu.M, 25 .mu.M, 30 .mu.M,
40 .mu.M, or 50 .mu.M, although the concentration is not limited
thereto. The concentration is preferably 10 .mu.M.
[0089] Concerning culture conditions, culture is conducted at about
30.degree. C. to 40.degree. C., and preferably at about 37.degree.
C., although the temperature is not limited thereto. Culture is
conducted under an atmosphere of air containing CO.sub.2, and the
CO.sub.2 concentration is preferably about 2% to 5%.
[0090] The culture period is not particularly limited because
long-term culture would not cause any problems. For example, the
culture period is at least 3 days, 4 days, 5 days, 6 days, 7 days,
8 days, 9 days, 10 days, 11 days, or 12 days. The culture period is
preferably at least 6 days, and it is particularly preferably 6
days. When the ROCK inhibitor is added, the duration of addition is
1 day or 2 days, and preferably 2 days. When the HDAC inhibitor is
further added, such addition is initiated on the day following the
initiation of the step, and culture is conducted for at least 3
days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or
11 days. Culture is preferably conducted for at least 5 days, and
particularly preferably for 5 days, in the presence of the HDAC
inhibitor.
(2) Step of Culture in a Medium Containing a BMP Inhibitor and a
TGF.beta. Inhibitor (Step 2)
[0091] A medium used in this step can be prepared from a medium
used for animal cell culture as a basal medium. Examples of basal
media include IMDM medium, Medium 199, Eagle's Minimum Essential
Medium (EMEM), .alpha.MEM medium, Dulbecco's modified Eagle's
Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's
medium, NEUROBASAL.RTM. Medium (Life Technologies), and a mixture
of any such media. A medium may or may not contain blood serum. A
medium may optionally contain one or more serum substitutes
selected from among, for example, albumin, transferrin, Knockout
Serum Replacement (KSR) (an FBS serum substitute used for ES cell
culture), N2 supplements (Invitrogen), B27 supplements
(Invitrogen), fatty acid, insulin, collagen precursors, trace
elements, 2-mercaptoethanol, and 3'-thiol glycerol. In addition, a
medium can contain one or more substances selected from among, for
example, lipids, amino acids, L-glutamine, GLUTAMAX.TM.
(Invitrogen), nonessential amino acids, vitamins, growth factors,
low-molecular-weight compounds, antibiotics, antioxidants, pyruvic
acids, buffer agents, and inorganic salts. A medium mixture of DMEM
and Ham's F12 supplemented with GLUTAMAX.TM., B27, N2, 3'-thiol
glycerol, and ascorbic acid is preferable.
[0092] In this step, the cells obtained in the previous step (i.e.,
the step of pluripotent stem cell culture in a medium containing
activin A and a GSK3.beta. inhibitor) are cultured in a medium
prepared by supplementing the basal medium with a BMP inhibitor and
a TGF.beta. inhibitor.
[0093] Examples of BMP inhibitors include: protein-based
inhibitors, such as Chordin, Noggin, and Follistatin; dorsomorphin
(i.e.,
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 (i.e.,
4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline).
Dorsomorphin and LDN-193189 are commercially available from
Sigma-Aldrich and Stemgent, respectively.
[0094] A BMP inhibitor that can be preferably used in the present
invention is Noggin. The Noggin concentration in a medium is not
particularly limited, provided that BMP can be inhibited. For
example, such concentration is 1 ng/ml to 2 .mu.g/ml, and it is
specifically 1 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml, 200 ng/ml, 300
ng/ml, 400 ng/ml, 500 ng/ml, 600 ng/ml, 700 ng/ml, 800 ng/ml, 900
ng/ml, 1 .mu.g/ml, or 2 .mu.g/ml. The concentration is preferably
100 ng/ml.
[0095] The term "TGF.beta. inhibitor" used herein refers to a
substance that inhibits signal transmission from the binding of
TGF.beta. to a receptor leading to SMAD. A TGF.beta. inhibitor is
not particularly limited, provided that such substance inhibits
TGF.beta. from binding to a receptor; i.e., the ALK family, or such
substance inhibits phosphorylation of SMAD caused by the ALK
family. Examples thereof include Lefty-1 (e.g., NCBI Accession Nos.
mouse NM_010094 and human NM_020997), SB431542
(4-(4-(benzo[d][1,3]dioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl)benzam-
ide), 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), and derivatives thereof.
[0096] A TGF.beta. inhibitor that can be preferably used in the
present invention is SB431542. The SB431542 concentration in a
medium is not particularly limited, provided that TGF.beta. is
inhibited. For example, such concentration is 1 .mu.M to 500 .mu.M,
and it is specifically 1 .mu.M, 2 .mu.M, 3 .mu.M, 4 .mu.M, 5 .mu.M,
6 .mu.M, 7 .mu.M, 8 .mu.M, 9 .mu.M, 10 .mu.M, 15 .mu.M, 20 .mu.M,
25 .mu.M, 30 .mu.M, 35 .mu.M, 40 .mu.M, 45 .mu.M, 50 .mu.M, 60
.mu.M, 70 .mu.M, 80 .mu.M, 90 .mu.M, 100 .mu.M, 200 .mu.M, 300
.mu.M, 400 .mu.M, or 500 .mu.M. The concentration is preferably 10
.mu.M.
[0097] In this step, culture may be conducted in a culture vessel
treated with a coating agent. Examples of coating agents include BD
MATRIGEL.RTM., collagen, gelatin, laminin, heparan sulfate
proteoglycan, entactin, and a combination of any thereof, with
MATRIGEL.RTM. being preferable.
[0098] This step may be implemented by exchanging the cell culture
medium obtained in the previous step (a culture solution) with the
medium described above (a culture solution). Alternatively, cells
may be detached and reseeded in a culture vessel. When cells are to
be detached, particular cells may be selected, and, for example,
SOX17- and/or FOXA2-positive cells may be selected and used in this
step. This method is preferably implemented by means of media
exchange.
[0099] When the step comprises a process of cell detachment, a ROCK
inhibitor may be added to a culture solution, so as to inhibit
pluripotent stem cell death caused by detachment.
[0100] Concerning culture conditions, culture is conducted at about
30.degree. C. to 40.degree. C., and preferably at about 37.degree.
C., although the temperature is not limited thereto. Culture is
conducted under an atmosphere of air containing CO.sub.2, and the
CO.sub.2 concentration is preferably about 2% to 5%.
[0101] The culture period is not particularly limited because
long-term culture would not cause any problems. For example, the
culture period is at least 1 day, 2 days, 3 days, 4 days, 5 days, 6
days, 7 days, or 8 days. The culture period is preferably 4
days.
(3) Step of Culture in a Medium Containing BMP4, Retinoic Acid, and
a GSK3.beta. Inhibitor (Step 3)
[0102] A medium used in this step can be prepared from a medium
used for animal cell culture as a basal medium. Examples of basal
media include IMDM medium, Medium 199, Eagle's Minimum Essential
Medium (EMEM), .alpha.MEM medium, Dulbecco's modified Eagle's
Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's
medium, NEUROBASAL.RTM. Medium (Life Technologies), and a mixture
of any such media. A medium may or may not contain blood serum. A
medium may optionally contain one or more serum substitutes
selected from among, for example, albumin, transferrin, Knockout
Serum Replacement (KSR) (an FBS serum substitute used for ES cell
culture), N2 supplements (Invitrogen), B27 supplements
(Invitrogen), fatty acid, insulin, collagen precursors, trace
elements, 2-mercaptoethanol, and 3'-thiol glycerol. In addition, a
medium can contain one or more substances selected from among, for
example, lipids, amino acids, L-glutamine, GLUTAMAX.TM.
(Invitrogen), nonessential amino acids, vitamins, growth factors,
low-molecular-weight compounds, antibiotics, antioxidants, pyruvic
acids, buffer agents, and inorganic salts. A medium mixture of DMEM
and Ham's F12 supplemented with GLUTAMAX.TM., B27, N2, 3'-thiol
glycerol, and ascorbic acid is preferable.
[0103] In this step, the cells obtained in the previous step (i.e.,
the step of culture in a medium containing a BMP inhibitor and a
TGF.beta. inhibitor) are cultured in a medium prepared by
supplementing the basal medium with BMP4, retinoic acid, and a
GSK3.beta. inhibitor.
[0104] The term "BMP4" used herein refers to a protein encoded by
the polynucleotide shown in the NCBI Accession Number NM_001202,
NM_130850, or NM_130851, and it may be in an active form resulting
from cleavage by a protease.
[0105] The BMP4 concentration in a culture solution is not
particularly limited. For example, such concentration is 10 ng/ml
to 1 .mu.g/ml, and it is specifically 10 ng/ml, 20 ng/ml, 30 ng/ml,
40 ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100
ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 600 ng/ml, 700
ng/ml, 800 ng/ml, 900 ng/ml, or 1 .mu.g/ml. The concentration is
preferably 20 ng/ml.
[0106] While all-trans retinoic acid (ATRA) is exemplified as
retinoic acid, artificially modified retinoic acid that retains
functions of naturally occurring retinoic acid may be used.
Examples thereof 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.,
U.S.A., 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.
[0107] The retinoic acid concentration in a medium is not
particularly limited. For example, such concentration is 1 nM to 1
.mu.M, and it is specifically 1 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25
nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200
nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, or 1
.mu.M. The concentration is preferably 50 nM to 1 .mu.M.
[0108] The GSK3.beta. inhibitor as described above can be used in
this step, and the GSK3.beta. inhibitor is preferably CHIR99021. In
this step, the CHIR99021 concentration in a medium is, for example,
1 nM to 50 .mu.M, and it is specifically 1 nM, 10 nM, 50 nM, 100
nM, 500 nM, 750 nM, 1 .mu.M, 1.5 .mu.M, 2 .mu.M, 2.5 .mu.M, 3
.mu.M, 3.5 .mu.M, 4 .mu.M, 4.5 .mu.M, 5 .mu.M, 6 .mu.M, 7 .mu.M, 8
.mu.M, 9 .mu.M, 10 .mu.M, 15 .mu.M, 20 .mu.M, 25 .mu.M, 30 .mu.M,
40 .mu.M, or 50 .mu.M, although the concentration is not limited
thereto. In this step, the concentration is preferably 1.5 .mu.M to
3.5 .mu.M.
[0109] In this step, culture may be conducted in a culture vessel
treated with a coating agent. A coating agent may be a naturally
occurring or artificially synthesized extracellular matrix.
Examples thereof include BD MATRIGEL.RTM., collagen, gelatin,
laminin, heparan sulfate proteoglycan, entactin, and a combination
of any thereof, with MATRIGEL.RTM. being preferable.
[0110] This step may be implemented by exchanging the cell culture
medium obtained in the previous step (a culture solution) with the
medium described above (a culture solution). Alternatively, cells
may be detached and reseeded in a culture vessel. When cells are to
be detached, particular cells may be selected, and, for example,
SOX2-, SOX17-, and/or FOXA2-positive cells may be selected and used
in this step. This method is preferably implemented by means of
media exchange.
[0111] When the step comprises a process of cell detachment, a ROCK
inhibitor may be added to a medium, so as to inhibit pluripotent
stem cell death caused by detachment.
[0112] Concerning culture conditions, culture is conducted at about
30.degree. C. to 40.degree. C., and preferably at about 37.degree.
C., although the temperature is not limited thereto. Culture is
conducted under an atmosphere of air containing CO.sub.2, and the
CO.sub.2 concentration is preferably about 2% to 5%.
[0113] The culture period is not particularly limited because
long-term culture would not cause any problems. For example, the
culture period is at least 1 day, 2 days, 3 days, 4 days, 5 days, 6
days, 7 days, or 8 days. The culture period is preferably at least
4 days, and more preferably 4 days.
(4) Step of Ventral Anterior Foregut Cell Culture in a Medium
Containing a GSK3.beta. Inhibitor, FGF10, KGF, and a NOTCH Signal
Inhibitor (Step 4)
[0114] A medium used in this step can be prepared from a medium
used for animal cell culture as a basal medium. Examples of basal
media include IMDM medium, Medium 199, Eagle's Minimum Essential
Medium (EMEM), .alpha.MEM medium, Dulbecco's modified Eagle's
Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's
medium, NEUROBASAL.RTM. Medium (Life Technologies), and a mixture
of any such media. A medium may or may not contain blood serum. A
medium may optionally contain one or more serum substitutes
selected from among, for example, albumin, transferrin, Knockout
Serum Replacement (KSR) (an FBS serum substitute used for ES cell
culture), N2 supplements (Invitrogen), B27 supplements
(Invitrogen), fatty acid, insulin, collagen precursors, trace
elements, 2-mercaptoethanol, and 3'-thiol glycerol. In addition, a
medium can contain one or more substances selected from among, for
example, lipids, amino acids, L-glutamine, GLUTAMAX.TM.
(Invitrogen), nonessential amino acids, vitamins, growth factors,
low-molecular-weight compounds, antibiotics, antioxidants, pyruvic
acids, buffer agents, and inorganic salts. A medium mixture of DMEM
and Ham's F12 supplemented with GLUTAMAX.TM., B27 supplement,
L-ascorbic acid, monothioglycerol, penicillin, and streptomycin is
preferable.
[0115] In this step, the ventral anterior foregut cells obtained in
the previous step (i.e., the step of culture in a medium containing
BMP4, retinoic acid, and a GSK3.beta. inhibitor) are cultured in a
medium prepared by supplementing the basal medium with a GSK3.beta.
inhibitor, FGF10, KGF, and a NOTCH signal inhibitor.
[0116] The GSK3.beta. inhibitor as described above can be used in
this step, and the GSK3.beta. inhibitor is preferably CHIR99021. In
this step, the CHIR99021 concentration in a medium is, for example,
1 nM to 50 .mu.M, and it is specifically 1 nM, 10 nM, 50 nM, 100
nM, 500 nM, 750 nM, 1 .mu.M, 1.5 .mu.M, 2 .mu.M, 2.5 .mu.M, 3
.mu.M, 3.5 .mu.M, 4 .mu.M, 4.5 .mu.M, 5 .mu.M, 6 .mu.M, 7 .mu.M, 8
.mu.M, 9 .mu.M, 10 .mu.M, 15 .mu.M, 20 .mu.M, 25 .mu.M, 30 .mu.M,
40 .mu.M, or 50 .mu.M, although the concentration is not limited
thereto. In this step, the concentration is preferably 3 .mu.M.
[0117] The term "FGF10" used herein refers to a protein encoded by
the polynucleotide shown in the NCBI Accession Number NM_004465,
and it may be in an active form resulting from cleavage by a
protease. Such FGF10 is commercially available from, for example,
Life Technologies or Wako.
[0118] The FGF10 concentration in a medium is not particularly
limited. For example, such concentration is 1 ng/ml to 1 .mu.g/ml,
and it is specifically 1 ng/ml, 5 ng/ml, 10 ng/ml, 20 ng/ml, 50
ng/ml, 100 ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, or 1
.mu.g/ml. The concentration is preferably 10 ng/ml.
[0119] The term "keratinocyte growth factor (KGF)" used herein
refers to a protein encoded by the polynucleotide shown in the NCBI
Accession Number NM_002009, and it may be in an active form
resulting from cleavage by a protease. Such KGF is commercially
available from, for example, Wako.
[0120] The KGF concentration in a medium is not particularly
limited. For example, such concentration is 1 ng/ml to 1 .mu.g/ml,
and it is specifically 1 ng/ml, 5 ng/ml, 10 ng/ml, 20 ng/ml, 50
ng/ml, 100 ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, or 1
.mu.g/ml. The concentration is preferably 10 ng/ml.
[0121] The term "NOTCH signal inhibitor" used herein refers to a
substance that inhibits a Notch signal. Examples thereof include
DAPT
(N-[2S-(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl-1,1-dimethylethyl
ester-glycine), DBZ
(N-[(1S)-2-[[(7S)-6,7-dihydro-5-methyl-6-oxo-5H-dibenz[b,d]azepin-7-yl]am-
ino]-1-methyl-2-oxoethyl]-3,5-difluorobenzeneacetamide), Compound E
(N-[(1S)-2-[[(3S)-2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepi-
n-3-yl]-amino]-1-methyl-2-oxoethyl]-3,5-difluorobenzeneacetamide),
FLI-06 (cyclohexyl
1,4,5,6,7,8-hexahydro-2,7,7-trimethyl-4-(4-nitrophenyl)-5-oxo-3-quinoline-
carboxylate), and LY411575
(N2-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N1-[(7S)-5-methyl-6-o-
xo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide).
[0122] A NOTCH signal inhibitor that can be preferably used in the
present invention is DAPT. The DAPT concentration in a medium is
not particularly limited, provided that a Notch signal is
inhibited. For example, such concentration is 1 nM to 50 .mu.M, and
it is specifically 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 750 nM, 1
.mu.M, 1.5 .mu.M, 2 .mu.M, 2.5 .mu.M, 3 .mu.M, 3.5 .mu.M, 4 .mu.M,
4.5 .mu.M, 5 .mu.M, 6 .mu.M, 7 .mu.M, 8 .mu.M, 9 .mu.M, 10 .mu.M,
15 .mu.M, 20 .mu.M, 25 .mu.M, 30 .mu.M, 40 .mu.M, or 50 .mu.M, and
it is preferably 20 .mu.M.
[0123] In this step, culture may be conducted in a culture vessel
treated with a coating agent. A coating agent may be a naturally
occurring or artificially synthesized extracellular matrix.
Examples thereof include BD MATRIGEL.RTM., collagen, gelatin,
laminin, heparan sulfate proteoglycan, entactin, and a combination
of any thereof, with MATRIGEL.RTM. being preferable.
[0124] This step may be implemented by exchanging the cell culture
medium obtained in the previous step (a culture solution) with the
medium described above (a culture solution). Alternatively, cells
may be detached and reseeded in a culture vessel. When cells are to
be detached, particular cells may be selected, and, for example,
NKX2-1-, GATA6-, and/or HOPX-positive cells may be selected and
used in this step. This method is preferably implemented by means
of media exchange.
[0125] When the step comprises a process of cell detachment, a ROCK
inhibitor may be added to a culture solution, so as to inhibit
ventral anterior foregut cell death caused by detachment.
[0126] Concerning culture conditions, culture is conducted at about
30.degree. C. to 40.degree. C., and preferably at about 37.degree.
C., although the temperature is not limited thereto. Culture is
conducted under an atmosphere of air containing CO.sub.2, and the
CO.sub.2 concentration is preferably about 2% to 5%.
[0127] The culture period is not particularly limited because
long-term culture would not cause any problems. For example, the
culture period is at least 2 days, 3 days, 4 days, 5 days, 6 days,
7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14
days. The culture period is preferably at least 7 days, and it is
more preferably 7 days.
[Step of Isolating (Selecting) Alveolar Epithelial Progenitor
Cells]
[0128] The method of the present invention can further comprise,
following Step (4), a step of isolating carboxypeptidase M
(CPM)-positive cells as alveolar epithelial progenitor cells. The
isolated alveolar epithelial progenitor cells can be used in Step
(5). The isolated alveolar epithelial progenitor cells may
constitute a cell population including alveolar epithelial
progenitor cells. Preferably, the alveolar epithelial progenitor
cells account for 50%, 60%, 70%, 80%, or 90% or more of the cell
population including alveolar epithelial progenitor cells.
[0129] Alveolar epithelial progenitor cells can be isolated with
the use of reagents having specific affinity to CPM. Examples of
reagents having specific affinity that can be used in the present
invention include antibodies, aptamers, peptides, and compounds
that specifically recognize the substances of interest, with
antibodies or fragments thereof being preferable.
[0130] Antibodies may be polyclonal or monoclonal antibodies.
Examples of antibody fragments include a part of an antibody (e.g.,
an Fab fragment) and a synthetic antibody fragment (e.g., a
single-stranded Fv fragment, ScFv).
[0131] In order to recognize or separate cells that express CPM,
reagents having relevant affinity may be bound or conjugated to
substances that enable detection, such as a fluorescent label, a
radioactive label, a chemoluminescent label, an enzyme, biotin, or
streptoavidin, or substances that enable isolation and extraction,
such as Protein A, Protein G, beads, or magnetic beads.
[0132] Alternatively, reagents having relevant affinity may be
indirectly labeled. For example, pre-labeled antibodies (secondary
antibodies) that specifically bind to the antibodies described
above may be used.
[0133] Alveolar epithelial progenitor cells can be isolated
(extracted) by, for example, a method comprising conjugating
particles to a reagent having relevant affinity in order to
precipitate the cells, a method involving the use of magnetic beads
to select the cells with the aid of magnetism (e.g., MACS), a
method involving the use of a cell sorter with the aid of a
fluorescent label (e.g., FACS), or a method involving the use of a
support upon which antibodies or the like are immobilized (e.g., a
cell enrichment column).
[Step of Subjecting Alveolar Epithelial Progenitor Cells to
Cryopreservation]
[0134] Following Step (4), the resulting alveolar epithelial
progenitor cells may be subjected to cryopreservation. Thereafter,
the cryopreserved alveolar epithelial progenitor cells may be
thawed when Step (5) is initiated.
[0135] Alveolar epithelial progenitor cells may be subjected to
cryopreservation by, for example, suspending alveolar epithelial
progenitor cells in a stock solution comprising dimethyl sulfoxide
(DMSO) and the medium for Step (4) at 1:4 to 20 (preferably 1:9),
injecting the suspension into a freezing vial, introducing the vial
into a cell-freezing container immediately, and freezing the
resultant in a deep freezer at -20.degree. C. to -150.degree. C.
(preferably at -80.degree. C.) slowly over a period of 4 to 48
hours (preferably 24 hours), followed by storage in a liquid
nitrogen tank.
[0136] The cryopreserved cells can be thawed by, for example,
suspending the cells with the immediate addition of the pre-heated
medium used in Step (4), subjecting the same to centrifugation at
300 to 1500 rpm (preferably at 900 rpm) for 1 to 15 minutes
(preferably 5 minutes), suction-removing the supernatant, and
suspending the resultant again in the medium used in Step (4).
(5) Step of Three-Dimensional Culture of Alveolar Epithelial
Progenitor Cells in a Medium Containing a Steroid Drug, a cAMP
Derivative, a Phosphodiesterase Inhibitor, and KGF (Step 5)
[0137] A medium used in this step can be prepared from a medium
used for animal cell culture as a basal medium. Examples of basal
media include IMDM medium, Medium 199, Eagle's Minimum Essential
Medium (EMEM), .alpha.MEM medium, Dulbecco's modified Eagle's
Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's
medium, NEUROBASAL.RTM. Medium (Life Technologies), and a mixture
of any such media. A medium may or may not contain blood serum. A
medium may optionally contain one or more serum substitutes
selected from among, for example, albumin, transferrin, Knockout
Serum Replacement (KSR) (an FBS serum substitute used for ES cell
culture), N2 supplements (Invitrogen), B27 supplements
(Invitrogen), fatty acid, insulin, ITS Premix, collagen precursors,
trace elements, 2-mercaptoethanol, and 3'-thiol glycerol. In
addition, a medium can contain one or more substances selected from
among, for example, lipids, amino acids, L-glutamine, GLUTAMAX.TM.
(Invitrogen), nonessential amino acids, vitamins, growth factors,
low-molecular-weight compounds, antibiotics, antioxidants, pyruvic
acids, buffer agents, and inorganic salts. Ham's F12 medium
containing BSA, HEPES, calcium chloride, ITS Premix, B27
supplements, penicillin, and streptomycin is preferable.
[0138] In this step, the alveolar epithelial progenitor cells
obtained in the previous step (i.e., the step of culture in a
medium containing a GSK3.beta. inhibitor, FGF10, KGF, and a NOTCH
signal inhibitor) are cultured in a medium prepared by
supplementing the basal medium with a steroid drug, a cAMP
derivative, a phosphodiesterase inhibitor, and KGF.
[0139] The term "steroid drug" used herein refers to a steroidal
anti-inflammatory drug, such as glucocorticoid or a synthetic
derivative thereof. Specific examples thereof include
hydrocortisone, hydrocortisone succinate, prednisolone,
methylprednisolone, methylprednisolone succinate, triamcinolone,
triamcinolone acetonide, dexamethasone, and betamethasone.
[0140] A steroid drug that can be preferably used in the present
invention is dexamethasone. The dexamethasone concentration in a
medium is not particularly limited. For example, such concentration
is 1 nM to 1 .mu.M, and it is specifically 1 nM, 5 nM, 10 nM, 20
nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200
nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, or 1
.mu.M. The concentration is preferably 50 nM.
[0141] The term "cAMP derivative" used herein refers to a compound
with a modified cyclic AMP substituent. Examples thereof include
cyclic adenosine monophosphate (cAMP), 8-bromo cyclic adenosine
monophosphate (8-Br-cAMP), 8-chloro-cyclic adenosine monophosphate
(8-Cl-cAMP), 8-(4-chlorophenylthio)cyclic adenosine monophosphate
(8-CPT-cAMP), and dibutyryl cyclic adenosine monophosphate
(DB-cAMP).
[0142] A cAMP derivative that can be preferably used in the present
invention is 8-Br-cAMP. The 8-Br-cAMP concentration in a medium is
not particularly limited. For example, such concentration is 1
.mu.M to 1 mM, and it is specifically 1 .mu.M, 5 .mu.M, 10 .mu.M,
20 .mu.M, 30 .mu.M, 40 .mu.M, 50 .mu.M, 60 .mu.M, 70 .mu.M, 80
.mu.M, 90 .mu.M, 100 .mu.M, 200 .mu.M, 300 .mu.M, 400 .mu.M, 500
.mu.M, 600 .mu.M, 700 .mu.M, 800 .mu.M, 900 .mu.M, or 1 mM. The
concentration is preferably 100 .mu.M.
[0143] The term "phosphodiesterase inhibitor" used herein refers to
a compound that inhibits phosphodiesterase (PDE), so as to increase
the cAMP or cGMP concentration in the cells. Examples thereof
include 1,3-dimethylxanthine,
6,7-dimethoxy-1-(3,4-dimethoxybenzyl)isoquinoline,
4-{[3',4'-(methylenedioxy)benzyl]amino}-6-methoxyquinazoline,
8-methoxymethyl-3-isobutyl-1-methylxanthine, and
3-isobutyl-1-methylxanthine (IBMX).
[0144] A phosphodiesterase inhibitor that can be preferably used in
the present invention is IBMX. The IBMX concentration in a medium
is not particularly limited. For example, such concentration is 1
.mu.M to 1 mM, and it is specifically 1 .mu.M, 5 .mu.M, 10 .mu.M,
20 .mu.M, 30 .mu.M, 40 .mu.M, 50 .mu.M, 60 .mu.M, 70 .mu.M, 80
.mu.M, 90 .mu.M, 100 .mu.M, 200 .mu.M, 300 .mu.M, 400 .mu.M, 500
.mu.M, 600 .mu.M, 700 .mu.M, 800 .mu.M, 900 .mu.M, or 1 mM. The
concentration is preferably 100 .mu.M.
[0145] The KGF described above can be used in this step. The KGF
concentration in a medium is not particularly limited. For example,
such concentration is 10 ng/ml to 1 .mu.g/ml, and it is
specifically 10 ng/ml, 20 ng/ml, 30 ng/ml, 40 ng/ml, 50 ng/ml, 60
ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml, 200 ng/ml, 300
ng/ml, 400 ng/ml, 500 ng/ml, 600 ng/ml, 700 ng/ml, 800 ng/ml, 900
ng/ml, or 1 .mu.g/ml. The concentration is preferably 10 ng/ml.
[0146] In this step, a ROCK inhibitor, such as Y-27632, may further
be added to the medium. The Y-27632 concentration in a medium is,
for example, 100 nM to 50 .mu.M, and it is specifically 100 nM, 500
nM, 750 nM, 1 .mu.M, 2 .mu.M, 3 .mu.M, 4 .mu.M, 5 .mu.M, 6 .mu.M, 7
.mu.M, 8 .mu.M, 9 .mu.M, 10 .mu.M, 15 .mu.M, 20 .mu.M, 25 .mu.M, 30
.mu.M, 40 .mu.M, or 50 .mu.M, although the concentration is not
limited thereto. The concentration is preferably 10 .mu.M.
[0147] In this step, alveolar epithelial progenitor cells are
subjected to three-dimensional culture for maturation. The term
"three-dimensional culture" used herein refers to float culture of
cells in the form of cell masses (i.e., spheroids).
Three-dimensional culture can be carried out with the use of, for
example, Cell Culture Inserts provided by BD.
[0148] Three-dimensional culture may be conducted in the presence
of other cell species. Examples of other cell species that may be
used include human pulmonary fibroblasts and human fetal pulmonary
fibroblasts. Such cells are commercially available from, for
example, American Type Culture Collection (ATCC) and DV Biologics.
Alveolar epithelial progenitor cells are mixed with other cell
species at a rate of, for example, 1:10 to 500, and preferably at
1:50. A cell density in a medium is, for example,
0.5.times.10.sup.6 cells to 1.times.10.sup.7 cells/ml, and
preferably 2.5.times.10.sup.6 cells/ml.
[0149] The medium used for three-dimensional culture may be
prepared with the addition of an extracellular matrix to the medium
described above. The ratio of the volume of the medium to the
volume of the extracellular matrix is, for example, 1:0.25 to 10,
and preferably 1:1. An extracellular matrix is a supramolecular
structure that exists outside the cell, and it may be a naturally
occurring or artificial (recombinant or peptide hydrogel)
structure. Examples thereof include substances, such as collagen,
proteoglycan, fibronectin, hyaluronic acid, tenascin, entactin,
elastin, fibrillin, and laminin, and fragments thereof. These
extracellular matrices may be used in combination. For example,
extracellular matrices may be prepared from cells such as Corning
MATRIGEL.RTM.. An example of an artificial structure is a laminin
fragment or Corning PuraMatrix.RTM..
[0150] Concerning culture conditions, culture is conducted at about
30.degree. C. to 40.degree. C., and preferably at about 37.degree.
C., although the temperature is not limited thereto. Culture is
conducted under an atmosphere of air containing CO.sub.2, and the
CO.sub.2 concentration is preferably about 2% to 5%.
[0151] The culture period is not particularly limited because
long-term culture would not cause any problems. For example, the
culture period is at least 5 days, 6 days, 7 days, 8 days, 9 days,
10 days, 11 days, 12 days, 13 days, or 14 days. The culture period
is preferably at least 14 days, and more preferably 14 days.
[Step of Isolating (Selecting) Type II Alveolar Epithelial
Cells]
[0152] The method of the present invention can further comprise,
following Step (5), a step of isolating type II alveolar epithelial
cells. In such step, cells positive for one or more type II
alveolar epithelial cell markers selected from the group consisting
of SFTPC (surfactant protein C), EpCAM (epithelial cell adhesion
molecule), and CEACAM6 (carcinoembryonic antigen-related cell
adhesion molecule 6) or cells positive for the type II alveolar
epithelial cell markers and staining of acidic fractions (e.g.,
lysosomes) in live cells with, for example, LysoTracker.RTM. may be
isolated as type II alveolar epithelial cells.
[0153] Type II alveolar epithelial cells can be isolated in
accordance with the method for isolating CPM-positive cells as
alveolar epithelial progenitor cells. The isolated type II alveolar
epithelial cells may constitute a cell population including type II
alveolar epithelial cells. Preferably, the type II alveolar
epithelial cells account for 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, or 90% or more of the cell population including type II
alveolar epithelial cells.
[Step of Subjecting Type II Alveolar Epithelial Cells to
Cryopreservation]
[0154] Following Step (5), the resulting type II alveolar
epithelial cells may be subjected to cryopreservation.
[0155] Type II alveolar epithelial cells may be subjected to
cryopreservation by, for example, suspending type II alveolar
epithelial cells in a stock solution comprising DMSO and the medium
for Step (5) at 1:4 to 20 (preferably 1:9), injecting the
suspension into a freezing vial, introducing the vial into a
cell-freezing container immediately, and freezing the resultant in
a deep freezer at -20.degree. C. to -150.degree. C. (preferably at
-80.degree. C.) slowly over a period of 4 to 48 hours (preferably
24 hours), followed by storage in a liquid nitrogen tank.
[0156] The cryopreserved cells can be thawed by, for example,
suspending the cells with the immediate addition of the pre-heated
medium used in Step (5), subjecting the same to centrifugation at
300 to 1500 rpm (preferably at 900 rpm) for 1 to 15 minutes
(preferably 5 minutes), suction-removing the supernatant, and
suspending the resultant again in the medium used in Step (5).
[Pluripotent Stem Cells]
[0157] Pluripotent stem cells that can be used in the present
invention are stem cells that have the potential to differentiate
into any types of cells existing in organisms (i.e., pluripotency)
and have the potential to grow. Examples thereof include embryonic
stem cells (ES cells), nuclear transfer-derived embryonic stem
cells from cloned embryos (nt ES cells), sperm stem cells (GS
cells), embryonic germ cells (EG cells), induced pluripotent stem
cells (iPS cells), and pluripotent cells derived from cultured
fibroblasts and myeloid stem cells (Muse cells). In the present
invention, the use of iPS cells or Muse cells is preferable because
cells of interest can be obtained without destroying embryos.
(A) Embryonic Stem Cells
[0158] ES cells are pluripotent stem cells having the potential to
grow through autoreproduction, and they are established from
embryoblasts of early embryos (e.g., blastocysts) of mammalians
such as humans or mice.
[0159] ES cells are embryo-derived stem cells originating from
embryoblasts of blastocysts, which are embryos after the 8-cell
stage and the morula stage of fertilized eggs. Such ES cells have
the potential to differentiate into any types of cells constituting
an adult; that is, so-called pluripotency, and the potential to
grow through autoreproduction. ES cells were discovered in mice in
1981 (M. J. Evans and M. H. Kaufman, 1981, Nature 292: 154-156).
Thereafter, ES cells of primates, such as humans and monkeys, were
also established (J. A. Thomson et al., 1998, Science 282:
1145-1147; J. A. Thomson et al., 1995, Proc. Natl. Acad. Sci.,
U.S.A., 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).
[0160] ES cells can be established by extracting embryoblasts from
blastocysts of fertilized eggs of target animals and culturing the
embryoblasts on fibroblast feeders. Cells can be maintained via
subculture with the use of a culture solution supplemented with
substances such as leukemia inhibitory factors (LIF) and basic
fibroblast growth factors (bFGF). Human and monkey ES cells can be
established and maintained by the methods described in, for
example, U.S. Pat. No. 5,843,780; Thomson J. A. et al., 1995, Proc.
Natl. Acad. Sci., U.S.A., 92: 7844-7848; Thomson, J. A. et al.,
1998, Science 282: 1145-1147; H. Suemori et al., 2006, Biochem.
Biophys. Res. Commun., 345: 926-932; M. Ueno et al., 2006, Proc.
Natl. Acad. Sci. U.S.A., 103: 9554-9559; H. Suemori et al., 2001,
Dev. Dyn., 222: 273-279; H. Kawasaki et al., 2002, Proc. Natl.
Acad. Sci. U.S.A., 99: 1580-1585; and Klimanskaya I et al., 2006,
Nature 444: 481-485.
[0161] Human ES cells can be maintained with the use of a medium
for ES cell production, such as a DMEM/F-12 medium supplemented
with 0.1 mM 2-mercaptoethanol, 0.1 mM nonessential amino acids, 2
mM L-glutamic acid, 20% KSR, and 4 ng/ml bFGF, at 37.degree. C. in
the presence of 5% CO.sub.2 in a moist atmosphere (H. Suemori et
al., 2006, Biochem. Biophys. Res. Commun., 345: 926-932). It is
necessary that ES cells be subjected to subculture every 3 or 4
days. Subculture can be carried out with the use of, for example,
0.25% trypsin and 0.1 mg/ml collagenase IV in PBS containing 1 mM
CaCl.sub.2 and 20% KSR.
[0162] In general, ES cells can be selected via real-time PCR using
the expression of a gene marker such as alkaline phosphatase,
Oct-3/4, or Nanog as an indicator. When human ES cells are to be
selected, in particular, the expression of a gene marker such as
OCT-3/4, NANOG, or ECAD can be employed as an indicator (E. Kroon
et al., 2008, Nat. Biotechnol., 26: 443-452).
[0163] Human ES cells (e.g., WA01 (H1) and WA09 (H9)) are available
from the WiCell Research Institute, and KhES-1, KhES-2, and KhES-3
are available from the Institute for Frontier Medical Sciences,
Kyoto University (Kyoto, Japan).
(B) Sperm Stem Cells
[0164] Sperm stem cells are testis-derived pluripotent stem cells
that serve as sources for spermatogenesis. As with the case of ES
cells, sperm stem cells can be differentiated into various types of
cells. For example, sperm stem cells may be implanted into mouse
blastocysts, so that chimeric mice may be produced (M.
Kanatsu-Shinohara et al., 2003, Biol. Reprod., 69: 12-616; K.
Shinohara et al., 2004, Cell, 119: 1001-1012). Sperm stem cells are
capable of autoreproduction in a medium containing glial cell
line-derived neurotrophic factors (GDNF). In addition, sperm stem
cells can be obtained by repeating subculture under the same
culture conditions as with those used for ES cells (Masanori
Takebayashi et al., 2008, Experimental Medicine, Vol. 26, No. 5
(extra edition), pp. 41-46, Yodosha, Tokyo, Japan).
(C) Embryonic Germ Cells
[0165] As with ES cells, embryonic germ cells are pluripotent cells
that are established from primordial germ cells during the prenatal
period. Embryonic germ cells can be established by culturing
primordial germ cells in the presence of substances such as LIF,
bFGF, or stem cell factors (Y. Matsui et al., 1992, Cell, 70:
841-847; J. L. Resnicket al., 1992, Nature, 359: 550-551).
(D) Induced Pluripotent Stem Cells
[0166] Induced pluripotent stem (iPS) cells can be prepared by
introducing particular reprogramming factors into somatic cells in
the form of DNA or proteins. iPS cells are artificial stem cells
derived from somatic cells that have substantially the same
properties as ES cells, such as pluripotency and the potential to
grow through autoreproduction (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). Reprogramming
factors may be composed of genes that are expressed specifically in
ES cells, gene products or non-cording RNA thereof, genes that play
key roles in maintenance of the undifferentiated state of ES cells,
gene products or non-coding RNA thereof, or low-molecular-weight
compounds. Examples of genes included in reprogramming factors
include Oct3/4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc,
N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1,
beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, and Glis1.
Such reprogramming factors may be used alone or in combination.
Examples of combinations of reprogramming factors are described in
WO 2007/069666, WO 2008/118820, WO 2009/007852, WO 2009/032194, WO
2009/058413, WO 2009/057831, WO 2009/075119, WO 2009/079007, WO
2009/091659, WO 2009/101084, WO 2009/101407, WO 2009/102983, WO
2009/114949, WO 2009/117439, WO 2009/126250, WO 2009/126251, WO
2009/126655, WO 2009/157593, WO 2010/009015, WO 2010/033906, WO
2010/033920, WO 2010/042800, WO 2010/050626, WO 2010/056831, WO
2010/068955, WO 2010/098419, WO 2010/102267, WO 2010/111409, WO
2010/111422, WO 2010/115050, WO 2010/124290, WO 2010/147395, WO
2010/147612, Huangfu, D. et al., 2008, Nat. Biotechnol., 26:
795-797, Shi, Y. et al., 2008, Cell Stem Cell, 2: 525-528, Eminli,
S. et al., 2008, Stem Cells, 26: 2467-2474, Huangfu, D. et al.,
2008, Nat. Biotechnol., 26: 1269-1275, Shi, Y. et al., 2008, Cell
Stem Cell, 3, 568-574, Zhao, Y. et al., 2008, Cell Stem Cell, 3:
475-479, Marson, A. 2008, Cell Stem Cell, 3, 132-135, Feng, B. et
al., 2009, Nat Cell Biol., 11: 197-203, R. L. Judson et al., 2009,
Nat. Biotech., 27: 459-461, Lyssiotis, C. A. et al., 2009, Proc.
Natl. Acad. Sci., U.S.A. 106: 8912-8917, Kim, J. B. et al., 2009,
Nature, 461: 649-643, Ichida, J. K. et al., 2009, Cell Stem Cell,
5: 491-503, Heng, J. C. et al., 2010, Cell Stem Cell, 6: 167-74,
Han, J. et al., 2010, Nature, 463: 1096-100, Mali, P. et al., 2010,
and Stem Cells, 28: 713-720, Maekawa, M. et al., 2011, Nature, 474:
225-9.
[0167] Factors that are used to enhance cell establishment
efficiency are within the scope of the reprogramming factors
described above. Examples thereof include: histone deacetylase
(HDAC) inhibitors, such as low-molecular-weight inhibitors,
including valproic acid (VPA), trichostatin A, sodium butyrate, MC
1293, and M344, and nucleic acid-based expression inhibitors,
including siRNAs and shRNAs against HDAC (e.g., HDAC1 siRNA
Smartpool.RTM. (Millipore) and HuSH 29 mer shRNA constructs against
HDAC1 (OriGene)); MEK inhibitors (e.g., PD184352, PD98059, U0126,
SL327, and PD0325901); glycogen synthase kinase-3 inhibitors (e.g.,
Bio and CHIR99021); DNA methyltransferase inhibitors (e.g.,
5-azacytidine); histone methyltransferase inhibitors (e.g.,
low-molecular-weight inhibitors, such as BIX-01294, and nucleic
acid-based expression inhibitors against Suv39h1, Suv39h2, SetDB1
and G9a, such as siRNAs and shRNAs); an L-channel calcium agonist
(e.g., Bayk8644); butyric acid, TGF.beta. inhibitor, and ALK5
inhibitor (e.g., LY364947, SB431542, 616453, and A-83-01); p53
inhibitors (e.g., siRNA and shRNA against p53); ARID3A inhibitors
(e.g., siRNA and shRNA against ARID3A), miRNA, such as miR-291-3p,
miR-294, miR-295, and mir-302, Wnt signaling (e.g., soluble Wnt3a),
neuro-peptide Y, prostaglandins (e.g., prostaglandin E2 and
prostaglandin J2), hTERT, SV40LT, UTF1, IRX6, GLIS1, PITX2, and
DMRTB1. Such factors used to enhance cell establishment efficiency
are not particularly distinguished from reprogramming factors
herein.
[0168] When reprogramming factors are in the form of proteins, for
example, they may be introduced into somatic cells by a technique
such as lipofection, fusion with cell-permeable peptides (e.g.,
HIV-derived TAT and polyarginine), or microinjection.
[0169] In contrast, reprogramming factors in the form of DNA can be
introduced into somatic cells by a technique involving the use of a
vector such as a virus, plasmid, or artificial chromosome vector,
lipofection, a technique involving the use of a liposome, or
microinjection, for example. Examples of virus vectors include
retrovirus vectors, lentivirus vectors (Cell, 126, pp. 663-676,
2006; Cell, 131, pp. 861-872, 2007; Science, 318, pp. 1917-1920,
2007), adenovirus vectors (Science, 322, 945-949, 2008),
adeno-associated virus vectors, and Sendai virus vectors (WO
2010/008054). Examples of artificial chromosome vectors include
human artificial chromosome (HAC) vectors, yeast artificial
chromosome (YAC) vectors, and bacterial artificial chromosome (BAC,
PAC) vectors. Plasmids for mammalian animal cells can be used
(Science, 322: 949-953, 2008). Vectors can comprise regulatory
sequences, such as promoters, enhancers, ribosome-binding
sequences, terminators, or polyadenylation sites, so that nuclear
reprogramming substances can express. In addition, vectors can
comprise selection marker sequences, such as drug tolerance genes
(e.g., kanamycin tolerance genes, ampicillin tolerance genes, and
puromycin tolerance genes), thymidine kinase genes, or diphtheria
toxin genes, and reporter gene sequences, such as green fluorescent
proteins (GFP), .beta.-glucuronidase (GUS), or FLAG, according to
need. The vector may comprise LoxP sequences in positions
downstream and upstream of a gene encoding a reprogramming factor
or a gene encoding a promoter and a reprogramming factor binding
thereto, so as to eliminate such gene after the vector is
introduced into somatic cells.
[0170] When reprogramming factors are in the form of RNA, for
example, they may be introduced into somatic cells by a technique
such as lipofection or microinjection. Alternatively, RNA
comprising 5-methylcytidine and pseudouridine (TriLink
Biotechnologies) incorporated therein may be used, so as to
suppress degradation (Warren L, 2010, Cell Stem Cell 7:
618-630).
[0171] Examples of culture media used for iPS cell induction
include DMEM containing 10% to 15% FBS, a DMEM/F12 or DME medium
(such medium may adequately contain, for example, LIF,
penicillin/streptomycin, puromycin, L-glutamine, nonessential amino
acids, and .beta.-mercaptoethanol), commercially available culture
media (e.g., a medium for mouse ES cell culture; TX-WES medium,
Thrombo X), a medium for primate ES cell culture (a medium for
primate ES/iPS cell culture, ReproCELL Incorporated), and a
serum-free medium (mTeSR, Stemcell Technology).
[0172] For example, somatic cells are brought into contact with
reprogramming factors in a 10% FBS-containing DMEM or DMEM/F12
medium, culture is conducted at 37.degree. C. in the presence of 5%
CO.sub.2 for about 4 to 7 days, and the cells are reseeded on
feeder cells (e.g., mitomycin C-treated STO cells or SNL cells).
Culture is reinitiated in a medium for bFGF-containing primate ES
cell culture about 10 days after the somatic cells are first
brought into contact with the reprogramming factors, and iPS-like
colonies can then be formed at least about 30 to 45 days after such
contact.
[0173] Alternatively, culture may be conducted in a 10%
FBS-containing DMEM medium (this medium can further contain LIF,
penicillin/streptomycin, puromycin, L-glutamine, nonessential amino
acids, .beta.-mercaptoethanol, or the like, according to need) on
feeder cells (e.g., mitomycin C-treated STO cells or SNL cells) at
37.degree. C. in the presence of 5% CO.sub.2, and ES-like colonies
can then be formed at least about 25 to 30 days later.
Alternatively, use of the somatic cells to be reprogrammed instead
of feeder cells is preferable (Takahashi K, et al., 2009, PLoS One,
4: e8067 or WO 2010/137746), or use of an extracellular matrix
(e.g., laminin-5 (WO 2009/123349) and Matrigel (BD)) is
preferable.
[0174] In addition, culture may be conducted with the use of a
serum-free medium (Sun, N. et al., 2009, Proc. Natl. Acad. Sci.,
U.S.A. 106: 15720-15725). In order to enhance cell establishment
efficiency, iPS cells may be established under low-oxygen
conditions (oxygen concentration of 0.1% to 15%) (Yoshida, Y. et
al., 2009, Cell Stem Cell, 5: 237-241 or WO 2010/013845).
[0175] During the culture, medium exchange is initiated 2 days
after the initiation of culture, and the medium is exchanged with a
fresh medium once a day. The number of somatic cells used for
nuclear reprogramming is not limited, and it is about
5.times.10.sup.3 to about 5.times.10.sup.6 cells per 100 cm.sup.2
of a culture dish.
[0176] iPS cells can be selected in accordance with the
configuration of the formed colonies. When drug tolerance genes
that express in association with genes that express upon
reprogramming of somatic cells (e.g., Oct3/4 and Nanog) are
introduced as marker genes, in contrast, culture can be conducted
in a medium containing corresponding drugs (i.e., a selection
medium). Thus, established iPS cells can be selected. When marker
genes are fluorescent protein genes, fluorescent microscopic
observation may be carried out. When marker genes are luminescent
enzyme genes, luminescent substrates may be added. When marker
genes are chromogenic enzyme genes, chromogenic substrates may be
added. Thus, iPS cells can be selected.
[0177] The term "somatic cells" used herein refers to any animal
cells except for germline cells or pluripotent cells such as egg
cells, oocytes, and ES cells (preferably mammalian animal cells,
including those of humans). Examples of somatic cells include, but
are not limited to, embryonic (fetal) somatic cells, neonatal
(fetal) somatic cells, and mature healthy or affected somatic
cells. Somatic cells may be primary-cultured cells, subcultured
cells, or established cells. Specific examples of somatic cells
include: (1) tissue stem cells, such as neural stem cells,
hematopoietic stem cells, mesenchymal stem cells, and dental pulp
stem cells (i.e., somatic stem cells); (2) tissue progenitor cells;
and (3) differentiated cells, such as lymphocytes, epidermic cells,
endothelial cells, muscle cells, fibroblasts (e.g., skin cells),
hair cells, hepatic cells, gastric mucosal cells, intestinal cells,
splenic cells, pancreatic cells (e.g., pancreatic exocrine cells),
brain cells, pneumocytes, nephrocytes, and adipocytes.
[0178] When iPS cells are used as materials for transplantation,
use of somatic cells having the same or substantially the same HLA
genotype as that of a recipient is preferable, so that rejection
would not occur. When HLA genotypes are "substantially the same,"
such HLA genotypes are concordant with each other to the extent
that an immunosuppressive agent is able to suppress immune
responses to the transplanted cells. For example, such somatic
cells have HLA genotypes exhibiting concordance in 3 loci; i.e.,
HLA-A, HLA-B, and HLA-DR, or in 4 loci; i.e., HLA-A, HLA-B, HLA-DR,
and HLA-C.
(E) Nuclear Transfer-Derived ES Cells from Cloned Embryos
[0179] "nt ES cells" are nuclear transfer-derived ES cells produced
from cloned embryos, and such ES cells have substantially the same
properties as fertilized egg-derived 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).
Specifically, nuclear transfer ES cells (i.e., nt ES cells) are ES
cells that are established from embryoblasts of blastocysts derived
from cloned embryos resulting from substitution of an unfertilized
egg nucleus with a somatic cell nucleus. nt ES cells are produced
by the technique of nuclear transfer (J. B. Cibelli et al., 1998,
Nature Biotechnol., 16: 642-646) in combination with the technique
of ES cell production (Kiyoka Wakayama et al., 2008, Experimental
Medicine, Vol. 25, No. 5 (extra edition), pp. 47-52). In the case
of nuclear transfer, somatic cell nuclei are injected into
enucleated unfertilized eggs of mammalian animals, and culture is
conducted for several hours. Thus, such cells can be
reprogrammed.
(F) Multilineage-Differentiating Stress Enduring Cells (Muse
Cells)
[0180] Muse cells are pluripotent stem cells produced by the method
described in WO 2011/007900. More specifically, Muse cells are
pluripotent cells that are obtained by treating fibroblasts or
myeloid interstitial cells with trypsin for a long period of time
(preferably for 8 hours or 16 hours) and conducting float culture.
Such cells are positive for SSEA-3 and CD105.
[Kit for Producing Type II Alveolar Epithelial Cells from
Pluripotent Stem Cells]
[0181] The present invention provides a kit for producing type II
alveolar epithelial cells from pluripotent stem cells. The kit may
comprise growth factors, compounds, a medium, extracellular
matrices, a cell detachment solution, and an agent for coating the
culture vessel as used for induction of differentiation. The kit
may further comprise documents and/or instructions describing the
procedure for the induction of differentiation.
[Method for Type II Alveolar Epithelial Cell Culture]
[0182] In accordance with Step (5) of the method for producing type
II alveolar epithelial cells from pluripotent stem cells according
to the present invention, the present invention relates to a method
for type II alveolar epithelial cell culture comprising a step of
three-dimensional culture of type II alveolar epithelial cells in a
medium containing a steroid drug, a cAMP derivative, a
phosphodiesterase inhibitor, and KGF. An ROCK inhibitor, such as
Y-27632, may further be added to the medium.
[0183] With the addition of the WNT signal inhibitor and/or IGF2 to
the medium, type II alveolar epithelial cells can be maintained
efficiently.
[0184] The term "WNT signal inhibitor" refers to a substance that
inhibits WNT signals. Examples thereof include protein inhibitors,
such as WIF1, and low-molecular-weight compounds, such as IWP2
(N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthien-
o[3,2-d] pyrimidin-2-yl)thio]-acetamide), IWR1
(4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-q-
uinolinyl-benzamide), and ICG-001
((6S,9aS)-hexahydro-6-[(4-hydroxyphenyl)methyl]-8-(1-naphthalenylmethyl)--
4,7-dioxo-N-(phenylmethyl)-2H-pyrazino[1,2-a]pyrimidine-1(6H)-carboxamide)-
.
[0185] The term "WIF1" used herein refers to a protein encoded by
the polynucleotide shown in the NCBI Accession Number NM_007191,
and it may be in an active form resulting from cleavage by a
protease. Such WIF1 is commercially available from, for example,
R&D Systems.
[0186] The term "IGF2" used herein refers to a protein encoded by
the polynucleotide shown in the NCBI Accession Number NM_000612,
and it may be in an active form resulting from cleavage by a
protease. Such IGF2 is commercially available from, for example,
R&D Systems and Life Technologies.
[0187] A WNT signal inhibitor that can be preferably used in the
present invention is WIF1.
[0188] The WIF1 concentration in a medium is, for example, 10 ng/ml
to 1 .mu.g/ml, and it is specifically 10 ng/ml, 20 ng/ml, 30 ng/ml,
40 ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100
ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 600 ng/ml, 700
ng/ml, 800 ng/ml, 900 ng/ml, or 1 .mu.g/ml, although the
concentration is not limited thereto. The concentration is
preferably 300 ng/ml.
[0189] The IGF2 concentration in a medium is, for example, 10 ng/ml
to 1 .mu.g/ml, and it is specifically 10 ng/ml, 20 ng/ml, 30 ng/ml,
40 ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100
ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 600 ng/ml, 700
ng/ml, 800 ng/ml, 900 ng/ml, or 1 .mu.g/ml, although the
concentration is not limited thereto. The concentration is
preferably 100 ng/ml.
[0190] Type II alveolar epithelial cells may be primary-cultured
cells isolated from, for example, tissue, or type II alveolar
epithelial cells may be produced by the method for producing type
II alveolar epithelial cells from pluripotent stem cells according
to the present invention.
[0191] The process of subculture carried out in this step can be
repeated at intervals of, for example, at least 14 days to 35 days,
specifically, at intervals of at least 14 days, 16 days, 18 days,
20 days, 21 days, 22 days, 24 days, 26 days, 28 days, 30 days, 32
days, 34 days, or 35 days, and, preferably, at intervals of 14
days.
[Kit for Type II Alveolar Epithelial Cell Culture]
[0192] The present invention provides a kit for type II alveolar
epithelial cell culture. The kit may comprise growth factors,
compounds, a medium, an extracellular matrix, a cell detachment
solution, and an agent for coating the culture vessel used for the
culture described above. The kit may further comprise documents
and/or instructions describing the procedure for the culture.
[Applications of Type II Alveolar Epithelial Cells Obtained in the
Present Invention]
[0193] The type II alveolar epithelial cells obtained in the
present invention can be used for large-scale screening of
candidate drugs in vitro when developing therapeutic agents for
intractable respiratory diseases, such as idiopathic pulmonary
fibrosis, hereditary interstitial lung disease, and congenital
pulmonary alveolar proteinosis.
[0194] The type II alveolar epithelial cells obtained in the
present invention can be administered to patients afflicted with
diseases that destroy the pulmonary alveolus in the form of
pharmaceutical preparations. The type II alveolar epithelial cells
are prepared into the form of a sheet, and the sheet may be applied
to the alveolar epithelium of a patient. Alternatively, the type II
alveolar epithelial cells may be suspended in physiological saline
or the like, and the suspension may then be directly implanted in
the pulmonary alveolus of the patient. Accordingly, the present
invention provides an agent for treatment of pulmonary alveolar
diseases comprising type II alveolar epithelial cells obtained from
pluripotent stem cells in the manner described above.
[0195] In the present invention, the number of type II alveolar
epithelial cells contained in the agent for treatment of pulmonary
alveolar diseases is not particularly limited, provided that the
transplanted grafts are able to survive after their administration.
The number of the cells may be adequately adjusted in accordance
with lesion size or body size.
[0196] Hereafter, the present invention is described in greater
detail with reference to the Examples, although the technical scope
of the present invention is not limited to these Examples.
EXAMPLE 1
Method for Inducing Type II Alveolar Epithelial Cells
1. Method for Inducing Type II Alveolar Epithelial Cells
[0197] FIG. 1 shows a method for inducing type II alveolar
epithelial cells from ventral anterior foregut cells using human
pluripotent stem cells.
1-1. Induction of Differentiation from Human Pluripotent Stem Cells
into Ventral Anterior Foregut Cells in Step 1-3
[0198] In accordance with the method described in Gotoh, S. et al.,
Stem Cell Reports, 2014, Vol. 3, pp. 394-403, human pluripotent
stem cells were induced to differentiate into ventral anterior
foregut cells.
[0199] Human iPS cells (201B7, 604A1) were provided by Professor
Yamanaka at Kyoto University, human ES cells (H9) were provided by
WiCell Research Institute, and the cells were cultured in
accordance with a conventional technique (Takahashi, K. et al.,
Cell, 131: 861-872, 2007; Okita, K., et al., Stem Cells, 31:
458-466, 2013; Gotoh, S., et al., Stem Cell Reports, 3: 394-403,
2014).
[0200] In accordance with the method described in Mae S., et al,
Nat. Commun., 4: 1367, 2013, according to a gene knock-in
technique, SFTPC-reporter 201B7 (i.e., (SFTPC-GFP reporter iPS cell
line B2-3) was produced by introducing an EGFP sequence into a site
downstream of the SFTPC initiation codon of the human iPS cells
(201B7).
[0201] The ventral anterior foregut cells were induced by detaching
human pluripotent stem cells with the use of Accutase, seeding the
cells in a 24-well plate coated with Matrigel at 2.0.times.10.sup.5
cells/well or in a 6-well plate coated with Matrigel at
9.6.times.10.sup.5 cells/well, and conducting culture under the
conditions described below.
1-1-1. Step 1
[0202] The seeded cells (Day 0) were cultured in a basal medium
(RPMI1640 (Nacalai Tesque) containing 2% B27 (Life Technologies)
and a 0.5% penicillin/streptomycin stock solution (Life
Technologies)) supplemented with 100 ng/ml activin A (R&D
Systems), 1 .mu.M CHIR99021, and 10 .mu.M Y-27632. On the following
day (Day 1), the medium was exchanged with the basal medium
containing 100 ng/ml activin A, 1 .mu.M CHIR99021, and 0.25 mM NaB,
the medium was exchanged with another medium under the same
conditions on the following day (Day 2) and 3 days later (Day 4),
and culture was conducted for 5 days.
[0203] Alternatively, the seeded cells (Day 0) were cultured in the
basal medium supplemented with 100 ng/ml activin A, 1 .mu.M
CHIR99021, and 10 .mu.M Y-27632. On the following day (Day 1), the
medium was exchanged with the basal medium containing 100 ng/ml
activin A, 1 .mu.M CHIR99021, 10 .mu.M Y-27632, and 0.125 mM or
0.25 mM NaB. On the following day (Day 2), the medium was exchanged
with the basal medium containing 100 ng/ml activin A, 1 .mu.M
CHIR99021, and 0.125 mM or 0.25 mM NaB. The medium was then
exchanged with another medium of the same conditions 3 days after
the initiation of culture (Day 4).
1-1-2. Step 2
[0204] The cells obtained in Step 1 (Day 6) were cultured in a
basal medium (DMEM/F12 medium (Life Technologies) containing 1%
GLUTAMAX.TM. supplement (Life Technologies), 2% B27 supplement, 1%
N2 supplement (Life Technologies), 0.8% StemSure.TM. 50 mmol/l
monothioglycerol solution (Wako), 50 .mu.g/ml L-ascorbic acid
(Sigma Aldrich), and 0.5% penicillin/streptomycin stock solution)
supplemented with 100 ng/ml hNoggin (R&D Systems) and 10 .mu.M
SB-431542 for 4 days. In this case, the medium was exchanged with
another medium under the same conditions every other day.
1-1-3. Step 3
[0205] The cells obtained in Step 2 (Day 10) were cultured in the
basal medium used in Step 2 containing 20 ng/ml hBMP4 (HumanZyme,
Inc.), 0.05 .mu.M, 0.5 .mu.M, or 1.0 .mu.M all-trans retinoic acid
(ATRA), and 2.5 .mu.M or 3.5 .mu.M CHIR99021 for 4 days. In this
case, the medium was exchanged with another medium under the same
conditions every other day.
1-2. Two-Dimensional Culture in Step 4
[0206] The ventral anterior foregut cells on Day 14 (upon
completion of Step 3) induced to differentiate in Section 1-1 above
were cultured in the medium for Step 4 for 7 days and the alveolar
epithelial progenitor cells were thus obtained efficiently. The
composition of the medium for Step 4 was composed of a basal medium
comprising DMEM/F12 (Life Technologies), 1.times. GLUTAMAX.TM.
(Life Technologies), 1.times. B27 supplement (Life Technologies),
0.05 mg/ml L-ascorbic acid (Sigma-Aldrich), 0.4 mM monothioglycerol
(Wako), and 50 U/ml penicillin/streptomycin (Life Technologies)
supplemented with 3 .mu.M CHIR99021, 10 ng/ml FGF10, 10 ng/ml KGF
(Prospec), and 20 DAPT.
1-3. Isolation of Alveolar Epithelial Progenitor Cells via FACS
[0207] On Day 21 (upon completion of Step 4) in Section 1-2 above,
the alveolar epithelial progenitor cells were isolated with the use
of antibodies reacting with CPM via fluorescence activated cell
sorting (FACS). Y-27632 (10 .mu.M) was added to the medium 1 hour
before the alveolar epithelial progenitor cells were peeled.
Thereafter, the culture plate was washed with PBS (Nacalai Tesque),
and 0.5 mM EDTA/PBS was added, followed by incubation at 37.degree.
C. for 12 minutes. After EDTA/PBS was removed, Accutase (Innovative
Cell Technologies) was added, incubation was carried out at
37.degree. C. for 25 minutes, a DMEM medium (Nacalai Tesque)
supplemented with 2% FBS (Life Technologies) was added, and the
cells were then recovered via pipetting. The recovered cell
suspension was allowed to pass through a 40 .mu.m cell strainer
mesh (BD Falcon), and the resultant was centrifuged at 800 rpm for
5 minutes, followed by washing with 1% BSA/PBS. The mouse
anti-human CPM antibody (Leica Microsystems) was added as the
primary antibody, and the reaction was then allowed to proceed at
4.degree. C. for 15 minutes.
[0208] After the completion of primary antibody treatment, the
cells were washed 2 times with 1% BSA/PBS, the ALEXA FLUOR.RTM.
647-labeled anti-mouse IgG antibody (Life Technologies) was added
as the secondary antibody, and the reaction was then allowed to
proceed in the dark at 4.degree. C. for 15 minutes. After the
completion of secondary antibody treatment, the cells were washed 2
times with 1% BSA/PBS, and propidium iodide was added in the end.
Thereafter, CPM-positive cells and CPM-negative cells were isolated
via FACS with the use of BD FACSAria.RTM. II or FacsAria.RTM. III
(BD Biosciences), and the CPM-positive cells were used as the
alveolar epithelial progenitor cells. As the isotype control, cells
treated with mouse IgG1 (Sigma-Aldrich) were used for FACS
gating.
1-4. Three-Dimensional Coculture in Step 5
[0209] The alveolar epithelial progenitor cells isolated via FACS
or MACS in Step 4 or the cryopreserved alveolar epithelial
progenitor cells were mixed with the human fetal lung fibroblasts
(17.5-weeks pregnant, DV Biologies) at a ratio of 2:100, the cell
suspension comprising the cells at a cell density of
2.5.times.10.sup.6 cells/ml and the medium for Step 5 supplemented
with Y-27632 at the final concentration of 10 .mu.M was mixed with
MATRIGEL.RTM. (Corning) at a ratio of 1:1 at a low temperature, the
resultant was immediately added to the upper layer of the Cell
Culture Inserts, and the medium for Step 5 was added to the lower
layer. At this time, the amount to be added to the upper layer of
the 12-well plate Cell Culture Inserts is preferably 200 to 400
.mu.l, and the amount to be added to the upper layer of the 24-well
plate Cell Culture Inserts is preferably 100 .mu.l. The amount of
the medium for Step 5 to be added to the lower layer of the 12-well
plate Cell Culture Inserts is preferably 1 ml, and the amount
thereof to be added to the lower layer of the 24-well plate Cell
Culture Inserts is preferably 500 .mu.l.
[0210] On the first 2 days, Y-27632 was added to the medium for
Step 5 to the final concentration of 10 .mu.M. Thereafter, the
medium for Step 5 of the lower layer was selectively exchanged with
another medium every other day.
[0211] The medium for Step 5 was prepared with the addition of 50
nM dexamethasone (Sigma-Aldrich), 100 .mu.M 8-Br-cAMP (Biolog), 100
.mu.M IBMX (Wako), and 10 ng/ml KGF (Prospec) to the basal medium
composed of Hams' F12 (Wako), 0.25% BSA (Life Technologies), 15 mM
HEPES (Sigma-Aldrich), 0.8 mM CaCl.sub.2 (Nacalai Tesque), 1.times.
ITS Premix (Corning), 0.5.times. B27 Supplement (Life
Technologies), and 50 U/ml penicillin/streptomycin (Life
Technologies).
2. Results of Induction of Type II Alveolar Epithelial Cells
[0212] FIG. 2 shows the results of examination of the composition
of the medium for Step 4 shown in FIG. 1 in terms of the efficiency
for alveolar epithelial progenitor cell induction and isolation
using NKX2.1 as a marker protein.
[0213] FIG. 2A to FIG. 2D demonstrate as follows.
[0214] FIG. 2A shows 6 conditions under which examinations were
carried out.
[0215] FIG. 2B shows fluorescent immunostaining images of
NKX2.1.sup.+ cells on Day 21 (i.e., upon completion of Step 4)
after ventral anterior foregut cells were cultured under each
condition for 7 days. The highest NKX2.1-positive rate was achieved
under the condition (4).
[0216] FIG. 2C shows the results of flow cytometry of alveolar
epithelial progenitor cells cultured under the condition (4) on Day
21 (i.e., upon completion of Step 4) with the use of the anti-CPM
antibody. The results indicate that the human iPS cell lines
(201B7, 604A1) and the human ES cell line (H9) contain large
quantities of CPM.sup.+ cells.
[0217] FIG. 2D shows the results of nuclear staining of CPM.sup.+
cells and CPM.sup.- cells isolated from the cell lines shown in
FIG. 2C and allowed to adhere to glass slides via cytospinning and
fluorescent immunostaining of NKX2.1 carried out simultaneously
therewith. The results demonstrate that most NKX2.1.sup.+ cells are
included in CPM.sup.+ cells and NKX2.1.sup.- cells are not
substantially included in CPM.sup.- cells. While the case of the
ventral anterior foregut cells on Day 14 (i.e., upon completion of
Step 3) was described in Gotoh, S. et al., Stem Cell Reports, 2014,
Vol. 3, pp. 394-403, it was confirmed that the same would apply to
the case on Day 21 (i.e., upon completion of Step 4).
[0218] FIG. 3 shows the results of examination of the composition
of the medium for Step 4 in terms of the expression level of SFTPC,
which is a marker protein for type II alveolar epithelial cells, on
Day 35 (i.e., upon completion of Step 5). The ventral anterior
foregut cells were cultured in the media under the 6 different
conditions shown in FIG. 2 for 7 days, CPM.sup.+ cells were
isolated on Day 21 (i.e., upon completion of Step 4), the cells
were subjected to three-dimensional coculture with human fetal lung
fibroblasts for 14 days, and the SFTPC expression levels were then
evaluated via quantitative RT-PCR on Day 35 (i.e., upon completion
of Step 5). The highest SFTPC expression level was observed under
the condition (4) among the 6 conditions.
[0219] FIG. 4 shows the results of examination of the culture
period under the conditions shown in FIG. 2 (4) in terms of the
expression level of SFTPC, which is a marker protein for type II
alveolar epithelial cells, on Day 35 (i.e., upon completion of Step
5). The results indicate that the SFTPC expression level was likely
to be high on Day 35 when the duration of Step 4 is 7 days.
[0220] FIG. 5 shows the results of whole well imaging demonstrating
a process comprising isolating CPM.sup.+ cells (alveolar epithelial
progenitor cells) separately from the control CPM.sup.- cells using
the SFTPC (SFTPC-GFP) reporter iPS cells on Day 21 (i.e., upon
completion of Step 4), subjecting the isolated cells to
three-dimensional coculture with human fetal lung fibroblasts, and
inducing type II alveolar epithelial cells from alveolar epithelial
progenitor cells over a period of 14 days. The results indicate
that both CPM.sup.+ cells and CPM.sup.- cells formed spheroids with
the elapse of time, and the spheroids were increased and enlarged.
While CPM.sup.+ cells became SFTPC-GFP-positive from Day 11 to Day
14, CPM.sup.- cells remained negative.
[0221] FIG. 6 shows photographs demonstrating the results of
observation of spheroids formed from CPM.sup.+ cells on Day 35
(i.e., upon completion of Step 5) in a high-power field. The
results demonstrate that SFTPC-GFP.sup.+ cells formed tubular
structures.
[0222] FIG. 7 shows the results of flow cytometry conducted on Day
35 (i.e., upon completion of Step 5) demonstrating that the
proportion of SFTPC-GFP.sup.+ cells (i.e., type II alveolar
epithelial cells) reached 50% of the CPM.sup.+ cell-derived
EpCAM.sup.+ cells. Among the CPM.sup.- cell-derived EpCAM.sup.+
cells, the percentage of SFTPC-GFP.sup.+ cells was 0%. As the
negative control, the human iPS cell line (201B7) in the state
before reporter cells were prepared was used.
[0223] FIG. 8 demonstrates that both the SFTPC and SFTPB expression
levels were increased by the method for type II alveolar epithelial
cell induction shown in FIG. 1 (the new protocol), compared with
the method described in Gotoh, S. et al., Stem Cell Reports, 2014,
Vol. 3, pp. 394-403 (the previously published protocol).
"CPM.sup.+" represents the gene expression of the three-dimensional
co-culture product derived from CPM.sup.+ cells and "CPM.sup.-"
represents the gene expression of the three-dimensional co-culture
product derived from CPM.sup.- cells.
[0224] FIG. 9 demonstrates that various marker proteins for type II
alveolar epithelial cells are expressed in a CPM.sup.+ cell-derived
three-dimensional culture product on Day 35 (i.e., upon completion
of Step 5), in comparison with the case of a CPM.sup.- cell-derived
three-dimensional culture product. The results demonstrate that
many marker genes specific to type II alveolar epithelial cells,
such as SFTPA and SFTPD as pulmonary surfactant proteins, DC-LAMP
and ABCA3 localized in lamellar bodies that are morphologically
characteristics of type II alveolar epithelial cells, and LPCAT1
known as an enzyme that synthesizes a lipid contained in a
surfactant were expressed in CPM.sup.+ cell-derived
three-dimensional culture products.
[0225] FIG. 10 shows double fluorescent immunostaining images of
spheroids on Day 35 (i.e., upon completion of Step 5). FIG. 10
shows that various surfactant proteins (i.e., SFTPA, SFTPB, SFTPC,
and SFTPD) were expressed in cells in tubular forms and that
DC-LAMP as a lamellar body marker was localized particularly on the
tubular side.
[0226] FIG. 11 shows transmission electron microscopic images of
spheroids on Day 35 (i.e., upon completion of Step 5). Many
lamellar bodies characteristics of type II alveolar epithelial
cells were formed (the photograph on the right) and multi-vesicular
bodies as progenitors thereof were also observed (the photograph on
the left).
EXAMPLE 2
Method for Type II Alveolar Epithelial Cell Culture
1. Three-Dimensional Subculture of Type II Alveolar Epithelial
Cells
[0227] FIG. 12 shows a method for culturing the type II alveolar
epithelial cells induced from human pluripotent stem cells via
subculture in a three-dimensional coculture system over a long
period of time.
[0228] On Day 35 of type II alveolar epithelial cell induction in
Example 1 (i.e., upon completion of Step 5), the cell mass composed
of fibroblasts and spheroids subjected to three-dimensional
coculture was removed from the Cell Culture Inserts. The removed
cell mass was cut into small pieces of approximately 1 mm with the
use of a clean surgical knife, the cells were recovered with the
addition of PBS, and centrifugation was carried out at 900 rpm for
5 minutes to remove the supernatant. 0.1% Trypsin/EDTA was added to
the precipitate and incubation was then carried out at 37.degree.
C. for 15 minutes while adequately repeating pipetting.
[0229] Subsequently, DMEM supplemented with 2% FBS was added to
neutralize trypsin, followed by centrifugation at 4.degree. C. and
900 rpm for 7 minutes. The supernatant was suction-removed, 1%
BSA/PBS was added to the remaining cell pellet, and the resultant
was centrifuged again at 4.degree. C. and 900 rpm for 5 minutes.
The cell pellet was suspended in 1% BSA/PBS, the mouse anti-human
Ep-CAM antibody (Santa Cruz) was added thereto, and the reaction
was allowed to proceed at 4.degree. C. for 20 minutes.
[0230] Subsequently, the cells were washed 2 times with 1% BSA/PBS,
the ALEXA FLUOR.RTM. 647-labeled anti-mouse antibody (Life
Technologies) was added thereto, and the reaction was allowed to
proceed at 4.degree. C. for 20 minutes. After the reaction product
was washed 2 times with 1% BSA/PBS, propidium iodide was added in
the end, and SFTPC-GFP-positive cells were isolated via FACS with
the use of BD FACSAria.RTM. II or Aria.RTM. III (BD Biosciences).
The isolated cells were used as the type II alveolar epithelial
cells (AT2-P0).
[0231] When type II alveolar epithelial cells were to be isolated
without the use of reporter cells, the ALEXA FLUOR.RTM. 647-labeled
anti-EpCAM antibody and LysoTracker.RTM. (Life Technologies) or a
labeled antibody (e.g., a PE-labeled anti-CEACAM6 antibody)
reacting with the known surface antigen of the type II alveolar
epithelial cells (i.e., the AT2-antigen) were used to isolate
EpCAM-positive, LysoTracker -positive, or AT2-antigen-positive
cells via FACS.
[0232] When type II alveolar epithelial cells were to be
subcultured, the type II alveolar epithelial cells were mixed with
human fetal lung fibroblasts immediately after isolation, or
cryopreserved type II alveolar epithelial cells were mixed with
human fetal lung fibroblasts at a ratio of 1:50, the cell
suspension of the medium for Step 5 comprising the cells at a cell
density of 2.5.times.10.sup.6 cells/ml and Y-27632 at the final
concentration of 10 .mu.M was mixed with MATRIGEL.RTM. (Corning) at
a ratio of 1:1 at a low temperature, the resultant was immediately
added to the upper layer of the Cell Culture Inserts, and the
medium for Step 5 was added to the lower layer. The amount to be
added to the upper layer of the 12-well plate Cell Culture Inserts
is preferably 200 to 400 and the amount to be added to the lower
layer thereof is preferably 1 ml.
[0233] On the first 2 days after the subculture, Y-27632 was added
to the medium for Step 5 to the final concentration of 10 .mu.M.
Thereafter, the medium for Step 5 of the lower layer was
selectively exchanged with another medium every other day. The type
II alveolar epithelial cells were isolated 14 days after the
initiation of culture, and culture was continued by repeating the
three-dimensional coculture with the human fetal lung fibroblasts.
As a subculture goes on to the following generation, the cells were
designated as AT2-P1, AT2-P2 . . . .
[0234] In the medium for Step 5 supplemented with WIF1 (R&D
Systems) or IGF2 (R&D Systems) at 300 ng/ml or 100 ng/ml,
respectively, type II alveolar epithelial cells were maintained
with higher efficiency, compared with culture in the medium
supplemented with EGF (Wako), NRG1.beta. (Peprotech), CHIR99021
(Axon Medchem) at 10 ng/ml, 20 ng/ml, 3 .mu.M, respectively.
2. Results of Three-Dimensional Subculture of Type II Alveolar
Epithelial Cells
[0235] As shown in FIG. 12, the type II alveolar epithelial cells
obtained in Step 5 of differentiating type II alveolar epithelial
cells from alveolar epithelial progenitor cells were defined as
"AT2-P0," isolated, and subjected to three-dimensional coculture.
It was thus confirmed that culture could be continued from AT2-P1
to AT2-P5 (Day 105).
[0236] FIG. 13 demonstrates that SFTPC could be maintained at a
certain positive rate via subculture of type II alveolar epithelial
cells in a three-dimensional coculture system.
[0237] FIG. 14 shows double fluorescent immunostaining images
demonstrating that the spheroids formed by the type II alveolar
epithelial cells subjected to subculture (AT2-P3) also expressed
DC-LAMP and SFTPC as with the case of spheroids at the AT2-P0
stage.
[0238] FIG. 15 demonstrates that type II alveolar epithelial cells
were isolated via induction of differentiation with the use of the
human iPS cell line (604A1) instead of SFTPC-GFP reporter cells up
to Day 35 (i.e., upon completion of Step 5) and with the use of the
anti-EpCAM antibody and LysoTracker.RTM. at the AT2-P0 stage.
[0239] FIG. 15A and FIG. 15B demonstrate as follows.
[0240] FIG. 15A shows the results of flow cytometry conducted after
dual staining with the use of the anti-EpCAM antibody and
LysoTracker.RTM. DND26.
[0241] FIG. 15B shows the results of nuclear staining of
EpCAM.sup.+ LysoTracker.RTM. cells and EpCAM.sup.+LysoTracker.RTM.
cells isolated and allowed to adhere to glass slides via
cytospinning while isolating and fluorescent immunostaining of
SFTPC carried out simultaneously therewith. The results demonstrate
that most SFTPC.sup.+ cells were included in
EpCAM.sup.+LysoTracker.RTM. cells and SFTPC.sup.+ cells were not
substantially included in EpCAM.sup.+ LysoTracker.RTM. cells.
[0242] FIG. 16 demonstrates that type II alveolar epithelial cells
were isolated via induction of differentiation with the use of the
human iPS cell line (604A1) instead of SFTPC-GFP reporter cells up
to Day 35 (i.e., upon completion of Step 5) as with the case shown
in FIG. 15 and with the use of the anti-CEACAM6 antibody at the
AT2-P0 stage.
[0243] FIG. 16A and FIG. 16B demonstrate as follows.
[0244] FIG. 16A shows the results of flow cytometric analysis,
following single staining with the use of the anti-CEACAM6
antibody.
[0245] FIG. 16B shows the results of nuclear staining of
CEACAM6.sup.+ cells and CEACAM6.sup.- cells isolated and allowed to
adhere to glass slides via cytospinning and fluorescent
immunostaining of SFTPC carried out simultaneously therewith. Not
so much as the case shown in FIG. 15, most SFTPC.sup.+ cells were
included in CEACAM6.sup.+ cells, and SFTPC.sup.+ cells were not
substantially included in CEACAM6.sup.- cells.
[0246] FIG. 17 demonstrates that type II alveolar epithelial cells
were efficiently maintained in the medium for Step 5 supplemented
with WIF1 (300 ng/ml) or IGF2 (100 ng/ml), when isolating AT2-P0
with the use of the SFTPC-GFP reporter cells and subjecting the
isolated cells to subculture as shown in FIG. 12. In the case of
the control experiment for cell maintenance in the medium for Step
5, the percentage of type II alveolar epithelial cells maintained
in AT2-P1 was 48.4% relative to the epithelial cell component (9.4%
of the whole including fibroblasts used for coculture). When
various additives (300 ng/ml WIF, 100 ng/ml IGF2, 10 ng/ml EGF, 20
ng/ml NRG1.beta., 3 .mu.M CHIR99021) were added, in contrast, the
percentages of type II alveolar epithelial cells maintained were
54.4%, 58.0%, 5.3%, 29.5%, and 23.8%, respectively, relative to the
epithelial cell component (20.2%, 16.6%, 1.2%, 11.3%, and 0.5%,
respectively, of the whole).
EXAMPLE 3
Method for Storage of Alveolar Epithelial Progenitor Cells or Type
II Alveolar Epithelial Cells
1. Method for Storage of Alveolar Epithelial Progenitor Cells or
Type II Alveolar Epithelial Cells
1-1. Isolation of Alveolar Epithelial Progenitor Cells Via MACS and
Cryopreservation Thereof
[0247] FIG. 18 shows a method for inducing type II alveolar
epithelial cells by cryopreserving human alveolar epithelial
progenitor cells on Day 21 (i.e., upon completion of Step 4) and
subjecting the resulting cells to three-dimensional coculture.
[0248] On Day 21 after the induction of type II alveolar epithelial
cells in Example 1 (i.e., upon completion of Step 4), alveolar
epithelial progenitor cells were isolated via magnetic activated
cell sorting (MACS) with the use of antibodies reacting with CPM
and then cryopreserved. In the same manner as with the isolation of
alveolar epithelial progenitor cells via FACS in Example 1, the
reaction was allowed to proceed to the treatment with the primary
antibody using the mouse anti-human CPM antibody, and the reaction
product was then washed 2 times with 1% BSA/PBS.
[0249] Thereafter, the microbead-labeled anti-mouse IgG1 antibody
(Miltenyi Biotech) was added as the secondary antibody, and the
reaction was allowed to proceed at 4.degree. C. for 15 minutes.
After the reaction product was washed 2 times with 0.5% BSA/PBS
supplemented with 2 mM EDTA, CPM-positive cells were isolated via
MACS using magnetic separation columns (Miltenyi Biotech), and MACS
was repeated 2 times, so as to enhance the purity.
[0250] When the isolated alveolar epithelial progenitor cells were
cryopreserved, 5.0.times.10.sup.5 cells were suspended in 500 .mu.l
of a stock solution comprising dimethyl sulfoxide (DMSO)
(Sigma-Aldrich) and the medium for Step 4 at a ratio of 1:9, the
suspension was injected into a freezing vial (Nalgene), the vial
was immediately introduced into a cell-freezing container
(Nalgene), and the resultant was slowly frozen in a deep freezer at
-80.degree. C. over a period of 24 hours, followed by storage in a
liquid nitrogen tank.
[0251] When the cryopreserved cells were to be thawed, a cell
suspension was prepared with the rapid addition of 10 ml of the
pre-heated medium for Step 4, the suspension was centrifuged at 900
rpm for 5 minutes, the supernatant was suction-removed, and the
resultant was suspended again in the medium for Step 4.
1-2. Cryopreservation of Type II Alveolar Epithelial Cells
[0252] FIG. 20 shows a method for type II alveolar epithelial cell
culture comprising cryopreserving type II alveolar epithelial cells
and subjecting the resulting cells to three-dimensional
coculture.
[0253] When the type II alveolar epithelial cells induced in
Example 1 were isolated and then cryopreserved, 2.0.times.10.sup.5
cells were suspended in 200 .mu.l of a stock solution comprising
DMSO and the medium for Step 5 at a ratio of 1:9, the suspension
was injected into a freezing vial (Nalgene), the vial was
immediately introduced into a cell-freezing container (Nalgene),
and the resultant was slowly frozen in a deep freezer at
-80.degree. C. over a period of 24 hours, followed by storage in a
liquid nitrogen tank.
[0254] When the cryopreserved cells were to be thawed, a cell
suspension was prepared with the rapid addition of 10 ml of the
pre-heated medium for Step 5, the suspension was centrifuged at 900
rpm for 5 minutes, the supernatant was suction-removed, and the
resultant was suspended again in the medium for Step 5.
2. Results of Storage of Alveolar Epithelial Progenitor Cells or
Type II Alveolar Epithelial Cells
2-1. Results of Storage of Alveolar Epithelial Progenitor Cells
[0255] FIG. 19 demonstrates that type II alveolar epithelial cells
were differentiated from the cryopreserved alveolar epithelial
progenitor cells with an efficiency of approximately 20% as a
result of the process shown in FIG. 18.
2-2. Results of Storage of Type II Alveolar Epithelial Cells
[0256] As shown in FIG. 20, type II alveolar epithelial cells were
cryopreserved, and three-dimensional coculture was carried out with
the use of the resulting cells. Thus, culture was carried out while
maintaining SFTPC with a constant positive rate.
INDUSTRIAL APPLICABILITY
[0257] According to the present invention, type II alveolar
epithelial cells can be efficiently produced from pluripotent stem
cells, and type II alveolar epithelial cells can be maintained.
[0258] All publications, patents, and patent applications cited
herein are incorporated herein by reference in their entirety.
* * * * *