U.S. patent application number 16/917464 was filed with the patent office on 2020-10-15 for method for producing na ve pluripotent stem cells.
This patent application is currently assigned to ONO PHARMACEUTICAL CO., LTD.. The applicant listed for this patent is KEIO UNIVERSITY, ONO PHARMACEUTICAL CO., LTD.. Invention is credited to Fumihiko Kisa, Hideyuki OKANO, Seiji Shiozawa.
Application Number | 20200325453 16/917464 |
Document ID | / |
Family ID | 1000004929452 |
Filed Date | 2020-10-15 |
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United States Patent
Application |
20200325453 |
Kind Code |
A1 |
OKANO; Hideyuki ; et
al. |
October 15, 2020 |
METHOD FOR PRODUCING NA VE PLURIPOTENT STEM CELLS
Abstract
To produce and/or maintain naive pluripotent stem cells capable
of highly expressing genes important for maintaining an
undifferentiated state, which could not be achieved by known
methods for producing pluripotent stem cells. The present invention
can produce naive pluripotent stem cells capable of maintaining an
undifferentiated state by introducing and allowing transient
expression of six genes (Oct3/4, Klf4, c-Myc, Sox2, Nanog, and
Klf2) among the so-called initializing factors, and further
performing culturing in a medium containing LIF, an MEK inhibitor,
a GSK3 inhibitor, a cAMP production promoter, a TGF-.beta.
inhibitor and a PKC inhibitor. Thus, the problem of the present
invention can be solved.
Inventors: |
OKANO; Hideyuki; (Tokyo,
JP) ; Shiozawa; Seiji; (Tokyo, JP) ; Kisa;
Fumihiko; (Ibaraki, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ONO PHARMACEUTICAL CO., LTD.
KEIO UNIVERSITY |
Osaka
Tokyo |
|
JP
JP |
|
|
Assignee: |
ONO PHARMACEUTICAL CO.,
LTD.
Osaka
JP
KEIO UNIVERSITY
Tokyo
JP
|
Family ID: |
1000004929452 |
Appl. No.: |
16/917464 |
Filed: |
June 30, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15558473 |
Sep 14, 2017 |
10738280 |
|
|
PCT/JP2016/058577 |
Mar 17, 2016 |
|
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16917464 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 5/10 20130101; C12N
15/09 20130101; C12N 2501/60 20130101; C12N 2501/605 20130101; C12N
2501/20 20130101; C12N 2501/602 20130101; C12N 2501/603 20130101;
C12N 2501/606 20130101; C12N 2501/01 20130101; C12N 5/0696
20130101 |
International
Class: |
C12N 5/074 20060101
C12N005/074; C12N 5/10 20060101 C12N005/10; C12N 15/09 20060101
C12N015/09 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 18, 2015 |
JP |
2015-054907 |
Claims
1. A method for producing a naive induced pluripotent stem cell,
comprising allowing transient expression of the following six
genes: Nanog, Klf2, Oct3/4, Klf4, c-Myc, and Sox2 in a somatic
cell.
2. The method for producing a naive induced pluripotent stem cell
according to claim 1, further comprising performing culturing in a
medium containing LIF, an MEK inhibitor, a GSK3 inhibitor, a cAMP
production promoter, a TGF-.beta. inhibitor and a PKC
inhibitor.
3. The method for producing a naive induced pluripotent stem cell
according to claim 1, wherein the somatic cell is a human-derived
somatic cell.
4. The method for producing a naive induced pluripotent stem cell
according to claim 2, wherein the MEK inhibitor is PD0325901, the
GSK3 inhibitor is CHIR99021, the cAMP production promoter is
Forskolin, the TGF-.beta. inhibitor is A83-01, and the PKC
inhibitor is Go6983.
5. The method for producing a naive induced pluripotent stem cell
according to claim 2, wherein the medium is an N2B27 medium.
6. A method for maintaining a naive pluripotent stem cell,
comprising culturing the naive pluripotent stem cell in a medium
containing LIF, an MEK inhibitor, a GSK3 inhibitor, a cAMP
production promoter, a TGF-.beta. inhibitor and a PKC
inhibitor.
7. The method for maintaining a naive pluripotent stem cell
according to claim 6, wherein the naive pluripotent stem cell is a
naive induced pluripotent stem cell or a naive embryonic stem
cell.
8. The method for maintaining a naive pluripotent stem cell
according to claim 6, wherein the naive pluripotent stem cell is a
naive human induced pluripotent stem cell or a naive human
embryonic stem cell.
9. The method for maintaining a naive pluripotent stem cell
according to claim 6, wherein the naive pluripotent stem cell is a
naive human induced pluripotent stem cell.
10. The method for maintaining a naive pluripotent stem cell
according to claim 6, wherein the MEK inhibitor is PD0325901, the
GSK3 inhibitor is CHIR99021, the cAMP production promoter is
Forskolin, the TGF-.beta. inhibitor is A83-01, and the PKC
inhibitor is Go6983.
11. The method for maintaining a naive pluripotent stem cell
according to claim 6, wherein the medium is an N2B27 medium.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional of U.S. application Ser.
No. 15/558,473 (allowed) filed Sep. 14, 2017, which is a National
Stage application under 35 U.S.C. .sctn. 371 of International
Application No. PCT/JP2016/058577, filed on Mar. 17, 2016, and
claims the benefit of Japanese Application No. 2015-054907, filed
on Mar. 18, 2015, the disclosures of which are incorporated herein
by reference in their entireties.
TECHNICAL FIELD
[0002] The present invention relates to a method for producing
and/or maintaining a naive-state pluripotent stem cells
(hereinafter, also abbreviated as a "method of the present
invention"). The present invention also relates to naive
pluripotent stem cells produced and/or maintained by the method of
the present invention and having excellent pluripotency.
BACKGROUND ART
[0003] In recent years, in the field of regeneration medicine, with
the advent of technology of induced pluripotent stem cells
(hereinafter, also abbreviated as "iPS cells"), clinical studies
toward practical use of iPS cells are rapidly progressing. For
example, clinical tests to age-related macular degeneration
patients are already carried out using retinal pigment epithelium
cells induced from human iPS cells. In addition, clinical tests
using nerve cells, chondrocytes, and the like, derived from human
iPS cells are planned to be carried out (see, Non-Patent Literature
1).
[0004] The iPS cells are one type of pluripotent stem cells. The
pluripotent stem cells include embryonic stem cells (hereinafter,
also abbreviated as ES cells), and Epiblast Stem cells, in addition
to the iPS cells. In recent years, these pluripotent stem cells
have been known to have different basic characteristics depending
on differences in animal species from which cells are derived or
types of pluripotent stem cells. Specifically, mouse ES cells and
mouse iPS cells are close to the early stage of development, can be
easily cultured or genetically engineered, and highly proliferate,
thus enabling differentiation to be induced efficiently. Such cells
are called naive cells. On the other hand, human ES cells, human
iPS cells, and the like, represent epiblast characteristics in a
highly-developed stage. Therefore, it is difficult to genetically
engineer the cells, and differentiation is biased toward any one
specific germ-layer among three germ-layers. Such cells are called
primed cells (see, Non-Patent Literature 2). Therefore, in
regeneration medicine etc., in order to practically use pluripotent
stem cells, a producing method capable of reliably obtaining
pluripotent stem cells having characteristics of the naive state
rather than characteristics of the primed state has been
demanded.
[0005] As a method for producing human iPS cells, a method of
introducing so-called Yamanaka four factors (Oct3/4 gene, Sox2
gene, Klf4 gene, and c-Myc gene) into somatic cells (see, Patent
Literature 1) has been known. However, the method had a problem
that only primed human iPS cells were able to be produced. To date,
in order to produce naive human iPS cells, techniques to induce iPS
cells derived from various animals to a naive state have been
developed. Attempt to further introduce other genes into the
above-mentioned Yamanaka four factors, and consideration of medium
conditions by adding various compounds, have been carried out. For
example, in a method of introducing two factors, Nanog and Klf2,
and adding LIF (Leukemia inhibitory factor), PD0325901 (MEK
inhibitor), CHIR99021 (GSK3 inhibitor) and Go6983 (PKC inhibitor)
into a medium (see Non-Patent Literature 3), expression of KLF4 and
TFCP2L1 which are known to be naive gene markers is increased, and,
therefore, obtaining of human iPS cells in a state close to the
naive state is reported.
[0006] However, the above-mentioned technology has a problem that
expression of ESRRB (Estrogen-related receptor beta) that is
considered to be a gene important for maintaining of a naive
undifferentiated state is hardly observed (see Non-Patent
Literatures 3 and 4), and the like.
PRIOR ART LITERATURES
Patent Literatures
[0007] Patent Literature 1: Japanese Patent Publication No.
4183742
Non-Patent Literatures
[0007] [0008] Non-Patent Literature 1: BioScience Trends, Vol. 7,
No. 3, pages 157-158, 2013 [0009] Non-Patent Literature 2: Cell
Stem Cell, Vol. 4, pages 487-492, 2009 [0010] Non-Patent Literature
3: Cell, Vol. 158, page 1254-1269, 2014 Non-Patent Literature 4:
Nature Cell Biology, Vol. 11, No. 2, page 197-203, 2009
SUMMARY OF INVENTION
Technical Problem
[0011] The problem of the present invention is to produce and/or
maintain pluripotent stem cells capable of high expression of genes
important for maintaining of a naive undifferentiated
potential.
Solution to Problem
[0012] In order to solve the above-mentioned problem, the inventors
of the present invention have extensively studied. As a result,
surprisingly, the inventors have found a method for producing
pluripotent stem cells capable of maintaining a naive
undifferentiated state by introducing six genes (Oct3/4, Klf4,
c-Myc, Sox2, Nanog, and Klf2) among the so-called initializing
factors to allow the genes to transiently express, and further
performing culturing in a medium containing LIF, an MEK inhibitor,
a GSK3 inhibitor, a cAMP production promoter, a TGF-.beta.
inhibitor, and a PKC inhibitor, and have completed the present
invention.
[0013] The present invention relates to: [1] a method for producing
naive pluripotent stem cells, the method including: allowing
transient expression of two types of genes: Nanog and Klf2 in
primed pluripotent stem cells, and performing culturing in a medium
containing LIF, an MEK inhibitor, a GSK3 inhibitor, a cAMP
production promoter, a TGF-.beta. inhibitor, and a PKC
inhibitor,
[0014] [2] the producing method described in the above [1], wherein
the primed pluripotent stem cells are primed induced pluripotent
stem cells or primed embryonic stem cells,
[0015] [3] the method for producing naive human pluripotent stem
cells described in the above [1] or [2], wherein the primed
pluripotent stem cells are primed human induced pluripotent stem
cells or primed human embryonic stem cells,
[0016] [4] the method for producing naive human induced pluripotent
stem cells described in any one of the above [1] to [3], wherein
the primed pluripotent stem cells are the primed human induced
pluripotent stem cells,
[0017] [5] the producing method described in any one of the above
[1] to [4], further including a step of allowing transient
expression of the following four genes: Oct3/4, Klf4, c-Myc, and
Sox2,
[0018] [6] the producing method described in any one of the above
[1] to [5], wherein the MEK inhibitor is PD0325901, the GSK3
inhibitor is CHIR99021, the cAMP production promoter is Forskolin,
the TGF-.beta. inhibitor is A83-01, and the PKC inhibitor is
Go6983,
[0019] [7] the producing method described in anyone of the above
[1] to [6], wherein the medium is an N2B27 medium,
[0020] [8] a method for producing naive pluripotent stem cells, the
method including a step of allowing transient expression of the
following six genes: Nanog, Klf2, Oct3/4, Klf4, c-Myc, and Sox2 in
primed pluripotent stem cells,
[0021] [9] the producing method described in the above [8], wherein
the primed pluripotent stem cells are primed induced pluripotent
stem cells or primed embryonic stem cells,
[0022] [10] the method for producing naive human pluripotent stem
cells described in the above [8] or [9], wherein the primed
pluripotent stem cells are primed human induced pluripotent stem
cells or primed human embryonic stem cells,
[0023] [11] the method for producing naive human induced
pluripotent stem cells described in any one of the above [8] to
[10], wherein the primed pluripotent stem cells are primed human
induced pluripotent stem cells,
[0024] [12] a method for producing naive induced pluripotent stem
cells, the method comprising a step of allowing transient
expression of the following six genes: Nanog, Klf2, Oct3/4, Klf4,
c-Myc, and Sox2 in somatic cells,
[0025] [13] the producing method described in the above [12],
further including allowing transient expression of six genes:
Oct3/4, Klf4, c-Myc, Sox2, Nanog, and Klf2 in somatic cells and
performing culturing in a medium containing LIF, an MEK inhibitor,
a GSK3 inhibitor, a cAMP production promoter, a TGF-.beta.
inhibitor and a PKC inhibitor,
[0026] [14] the method for producing naive human induced
pluripotent stem cells described in the above [12] or [13], wherein
the somatic cells are human-derived somatic cells,
[0027] [15] the producing method described in the above [13] or
[14], wherein the MEK inhibitor is PD0325901, the GSK3 inhibitor is
CHIR99021, the cAMP production promoter is Forskolin, the
TGF-.beta. inhibitor is A83-01, and the PKC inhibitor is
Go6983,
[0028] [16] the producing method described in anyone of the above
[13] to [15], wherein the medium is an N2B27 medium,
[0029] [17] a method for maintaining naive pluripotent stem cells,
the method comprising culturing the naive pluripotent stem cells in
a medium containing LIF, an MEK inhibitor, a GSK3 inhibitor, a cAMP
production promoter, a TGF-.beta. inhibitor and a PKC
inhibitor,
[0030] [18] the maintaining method described in the above [17],
wherein the naive pluripotent stem cells are naive induced
pluripotent stem cells or naive embryonic stem cells,
[0031] [19] the method for maintaining naive human pluripotent stem
cells described in the above [17] or [18], wherein the naive
pluripotent stem cells are naive human induced pluripotent stem
cells or naive human embryonic stem cells,
[0032] [20] the method for maintaining naive human induced
pluripotent stem cells described any one of in the above [17] to
[19], wherein the naive pluripotent stem cells are naive human
induced pluripotent stem cells,
[0033] [21] The maintaining method described in any one of the
above [17] to [20], wherein the MEK inhibitor is PD0325901, the
GSK3 inhibitor is CHIR99021, the cAMP production promoter is
Forskolin, the TGF-.beta. inhibitor is A83-01, and the PKC
inhibitor is Go6983,
[0034] [22] the maintaining method described in any one of the
above [17] to [21], wherein the medium is an N2B27 medium.
[0035] [23] a naive pluripotent stem cell produced by the method
described in any one of the above [1] to [16],
[0036] [24] a naive pluripotent stem cell maintained by the method
described in any one of the above [17] to [22], and the like.
Advantageous Effects of Invention
[0037] According to a method for producing pluripotent stem cells
cultured in specific conditions of the present invention, it is
possible to stably produce pluripotent stem cells maintaining
characteristics of the naive state and having pluripotency.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1 shows a gene expression vector used for gene
introduction.
[0039] FIG. 2A shows photographs of colonies of cells,
respectively. FIG. 2B shows expression amounts of genes in the
cells, respectively. In the drawings, DOX represents doxycycline,
P2 represents cells at passage 2, and P3 represents cells at
passage 3.
[0040] FIG. 3 shows expression amounts of ESRRB genes in 201B7
cells (passage 1) cultured in media, respectively. In the drawing,
KSR 2iLFA and KSR 2iLFA+Go6983 represent media shown in Table 1,
and DOX represents doxycycline.
[0041] FIG. 4A shows photographs of colonies of 201B7 cells
cultured in media (left: passage 2, right: passage 3). FIG. 4B
shows expression amounts of ESRRB genes in the 201B7 cells cultured
in media (left: passage 2, right: passage 3). In the drawings,
2iL+Go6983 and 2iLFA+Go6983 represent media represented in Table 1,
and DOX represents doxycycline.
[0042] FIG. 5 shows expression amounts of ESRRB genes in
combinations of reprogramming factors. In the drawing, 2F
represents a combination of a NANOG gene and a KLF2 gene, 4F
represents a combination of Yamanaka four factors (an Oct3/4 gene,
an Sox2 gene, a Klf4 gene, and a c-Myc gene), 6F represents an
NANOG gene, a KLF2 gene and Yamanaka four factors (an Oct3/4 gene,
an Sox2 gene, a Klf4 gene, and a c-Myc gene).
[0043] FIG. 6A shows results of cell staining (green: MAP2, blue:
nuclear staining by Hoechst) of each iPS cell neural
differentiation-induced by the SDIA method for 10 days. FIG. 6B
shows rates of colonies in which MAP2 positive cells are present on
day 10 of induction of differentiation of the iPS cells,
respectively.
[0044] FIG. 7A shows results of cell staining (green:
.beta.III-Tubulin, red: GFAP) of iPS cells, induced neural
differentiation by Neurosphere. FIG. 7B shows rates of
differentiation of the iPS cells into astrocyte and nerve cells,
respectively.
[0045] FIG. 8 shows stages of conversion to a naive state (P1 to
P3). P1 represents cells at passage 1, P2 represents cells at
passage 2, and P3 represents cells at passage 3.
[0046] FIG. 9 shows a gene expression vector used for gene
introduction in Example 5.
DESCRIPTION OF EMBODIMENTS
[0047] The present invention will be described in detail
hereinafter.
[0048] In the present invention, a method for producing naive
pluripotent stem cells is carried out according to the following
steps. In other words, the naive pluripotent stem cells are
produced through the steps including: step (i): a step of
introducing a certain gene into somatic cells or primed pluripotent
stem cells using a gene expression vector; step (ii): a step of
culturing the somatic cells or the primed pluripotent stem cells
into which the gene has been introduced in the step (i) in a prime
medium, adding doxycycline thereto, and then cloning a gene
expression cell line; step (iii): a step of allowing transient
expression of the gene introduced in the step (i) in the gene
expression cell line cloned in the step (ii), and, during the time,
performing culturing in a naive medium into which various compounds
were added.
[0049] Furthermore, in the present invention, a method for
maintaining naive pluripotent stem cells includes culturing and
subculturing naive pluripotent stem cells in a naive medium
containing various compounds.
[0050] In the present invention, the somatic cells are not
particularly limited, and any somatic cells can be used. For
example, in addition to somatic cells at an embryonic stage, mature
somatic cells may be used. Examples of the somatic cells include
differentiated cells including (1) tissue stem cells (somatic stem
cells) such as neural stem cells, hematopoietic stem cells,
mesenchymal stem cells, and dental pulp stem cells, (2) tissue
precursor cells, (3) fibroblasts (skin cells, etc.), epithelial
cells, liver cells, lymphocytes (T cells and B cells), endothelial
cells, muscular cells, hair cells, gastric mucosa cells, intestine
cells, splenic cells, pancreatic cells (pancreatic outer secretion
cells, etc.), brain cells, pneumocytes, nephrocytes, skin cells,
and the like. Preferable somatic cells are human-derived somatic
cells.
[0051] In the present invention, the pluripotent stem cells are not
particularly limited, and examples thereof include embryonic stem
cells (ES cells), nuclear-transfer embryonic stem (ntES) cells
derived from cloned embryo, germline stem cells (GS cell), epiblast
cells, embryonic germ cells (EG cells), multipotent germline stem
cells (mGS cells), and induced pluripotent stem cells (iPS cells).
Among them, ES cells, ntES cells, and iPS cells are preferable, ES
cells and iPS cells are more preferable, and iPS cells are
particularly preferable.
[0052] Herein, living bodies from which these somatic cells or
pluripotent stem cells are derived are not particularly limited,
and examples thereof include human, non-human animals (for example,
monkey, sheep, cow, horse, dog, cat, rabbit, rat, and mouse). A
preferable example is human.
[0053] In the present invention, the primed pluripotent stem cells
are not particularly limited, but they mean cells excluding mouse
ES cells and mouse iPS cells which are recognized to be naive cells
among the pluripotent stem cells. Example include ES cells and iPS
cells derived from human, monkey, pig, sheep, dog, and cow, as well
as epiblast stem cells derived from human and non-human animal
cells mentioned above. Preferable examples include human ES cells
and human iPS cells, and particularly preferable example is human
iPS cells.
[0054] In the present invention, the cell line of human ES cells to
be used as the primed pluripotent stem cells is not particularly
limited, and examples thereof include H1, H9, Shef6, khES-1,
khES-2, khES-3, khES-4, and khES-5.
[0055] In the present invention, the cell line of the human iPS
cells used as the primed pluripotent stem cells is not particularly
limited, and examples thereof include WD39 (derived from
fibroblast), aTKA4 (derived from T cells), 201B6, 201B7, 253G1, and
253G4. Among them, WD39 or 201B7 is preferable.
[0056] In the present invention, genes to be introduced into
somatic cells or primed pluripotent stem cells are not particularly
limited as long as they are factors that are known as initializing
factors (reprogramming factors). Examples thereof include Oct3/4,
Klf4, c-Myc, Sox2, Nanog, Klf2, L-Myc, N-Myc, Klf5, Lin28, Tert,
Fbx15, ERas, ECAT15-1, ECAT15-2, Tcl1, .beta.-Catenin, ECAT1, Esg1,
Dnmt3L, ECAT8, Gdf3, Sox15, Fthl17, Sall4, Rex1, UTF1, Stella,
Stat3, Grb2, Prdm14, Nr5a1, Nr5a2, and E-cadherin. Herein, optional
combination of two or more genes selected from the gene group can
be introduced. Among them, the combination of Oct3/4, Klf4, c-Myc,
Sox2, Nanog, and Klf2 is preferable. However, when the cells into
which genes are to be introduced are primed pluripotent stem cells,
since any of the above-mentioned initializing factors are
expressed, initializing factors excluding the expressed factors may
be introduced. When the cells are human ES cells or human iPS cells
as the initializing factors, for example, a combination of Nanog
and Klf2 is preferable, and a combination of Oct3/4, Klf4, c-Myc,
Sox2, Nanog, and Klf2 is furthermore preferable. Furthermore,
species of introducing cells are preferably the same as the species
of the cells into which genes are to be introduced. Examples of the
genes to be introduced into human-derived cells preferably include
human genes. Example of the genes to be introduced into
human-derived somatic cells, human ES cells, or human iPS cells
include preferably a combination of human Nanog (NANOG), and human
Klf2 (KLF2), and further preferably a combination of human Oct3/4
(OCT3/4), human Klf4 (KLF4), human c-Myc (c-MYC), human Sox2
(SOX2), human Nanog (NANOG), and human Klf2 (KLF2).
[0057] In the present invention, the gene expression vectors are
not particularly limited, and examples thereof include a virus
vector, a plasmid vector, an artificial chromosome vector, and a
transposon vector. Examples of the virus vector include a
retrovirus vector, an adenovirus vector, a Sendai virus vector, a
lentivirus vector, and an adeno-associated virus vector.
[0058] In the present invention, after somatic cells or primed
pluripotent stem cells are subjected to gene introduction, they may
be cultured together with feeder cells. The feeder cells are not
particularly limited, and examples thereof include mouse embryonic
fibroblast cells (MEF cells), mouse embryonic fibroblast line (STO
cells), and the like.
[0059] In the present invention, the prime medium is not
particularly limited, and examples thereof include a medium
prepared by combining any of additive components such as
replacement serum (KSR; Knockout.TM. Serum Replacement
(Invitrogen)), fetal bovine serum (FBS), non-essential amino acid
(NEAA), L-glutamine, 2-mercaptoethanol, antibiotics (for example,
streptomycin, penicillin, puromycin, and mitomycin), and bFGF
(basic Fibroblast Growth Factor), and adding any combination into a
basal medium that is preferably DMEM (Dulbecco Modified Eagle
medium), a mixer medium of DMEM and F12 (DMEM/F12=1:1), and
Knockout.TM. D-MEM (available from Invitrogen).
[0060] In the present invention, cloning refers to a method of
screening cells in which expression of genes is recognized in any
of the somatic cells or primed pluripotent stem cells into which
initializing factors have been gene-introduced. Examples of such
screening method include a method of verifying fluorescence of a
fluorescent protein gene which was incorporated into a gene
expression vector in advance under fluorescence microscope.
Examples of the fluorescence include a green fluorescent protein
(GFP) gene, yellow fluorescent protein (YFP) gene (for example,
Venus), a cyan fluorescent protein (CFP) gene (for example,
Cerulean), a red fluorescent protein (RFP) gene (for example,
TOMATO), and the like. Cells screened by this method is called a
gene expression cell line in the present invention.
[0061] In the present invention, various compounds in the step
(iii) and the maintaining method include a LIF (Leukemia inhibitory
factor), an MEK inhibitor, a GSK3 inhibitor, a cAMP production
promoter, a TGF-.beta. inhibitor, or a PKC inhibitor. It is
preferable that all of these compounds are added to a naive
medium.
[0062] In the present invention, the MEK inhibitor is not
particularly limited, and examples thereof include PD0325901
(N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)ami-
no]-benzamide; CAS registry number: 391210-10-9), U0126
(1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadien e; CAS
registry number: 109511-58-2), PD98059
(2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; CAS registry
number: 167869-21-8), PD184352 (2-(2-chloro-4-iodo
phenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide; CAS
registry number: 212631-79-3, and the like. Especially, PD0325901
is preferable.
[0063] In the present invention, the GSK3 inhibitor is not
particularly limited, and examples thereof include
CHIR99021(6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazole-2-yl)-2-
-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitr ile; CAS
registry number: 252917-06-9), BIO (6-bromoindirubin-3'-oxime; CAS
registry number: 667463-62-9), Kenpaullone
(9-bromo-7,12-dihydroindolo[3,2-d] [1]benzazepin-6(5H)-one; CAS
registry number: 142273-20-9), IM-16 (3-(4-fluorophenylethyl
amino)-1-methyl-4-(2-methyl-1H-indole-3-yl)-1H-pyrrole-2,5-dione;
CAS registry number: 1129669-05-1), and the like. Especially,
CHIR99021 is preferable.
[0064] In the present invention, the cAMP production promoter is
not particularly limited, and examples thereof include Forskolin
(CAS registry number: 66428-89-5), and the like.
[0065] In the present invention, the TGF-.beta. inhibitor is not
particularly limited, and examples thereof include A83-01
(3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carboth-
ioamide; CAS registry number: 909910-43-6), SB431542
(4-[4-(1,3-benzodioxole-5-yl)-5-(2-pyridinyl)-1H-imidazole-2-yl]-benzamid-
e; CAS registry number: 301836-41-9), and the like. Especially,
A83-01 is preferable.
[0066] In the present invention, the PKC inhibitor is not
particularly limited, and examples thereof include Go6983
(3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indole-3-yl]-4-(1H-indole-3--
yl)-1H-pyrrole-2,5-dione; CAS registry number: 133053-19-7),
GF109203X
(3-(1-(3-dimethylamino)propyl)-1H-indole-3-yl)-4-(1H-indole-3-yl)-1H-pyrr-
ole-2,5-dione; CAS registry number: 133052-90-1), and the like.
Especially, Go6983 is preferable.
[0067] In the present invention, the naive medium is not
particularly limited, and examples thereof include a medium
prepared by combining any of additive components such as a
non-essential amino acid (NEAA), L-glutamine, 2-mercaptoethanol,
antibiotics (for example, streptomycin, penicillin, puromycin, and
mitomycin), bovine serum albumin (BSA), and the like, and adding
any combination into a basal medium that is preferably a N2B27
medium (a medium obtained by mixing 1:1 of an N2 medium in which N2
supplement has been added to a DMEM/F12 medium and a B27 medium in
which B27 supplement has been added to a Neurobasal medium).
[0068] In the present invention, the concentration in the medium of
various compounds to be added to the naive medium is not
particularly limited. For example, it is preferable that the
concentration of LIF is 1 ng/mL to 100 ng/mL; the concentration of
an MEK inhibitor is 50 nM to 100 .mu.M; the concentration of a GSK3
inhibitor is 50 nM to 100 .mu.M; the concentration of a cAMP
production promoter is 50 nM to 100 .mu.M; the concentration of a
TGF-.beta. inhibitor is 10 nM to 100 .mu.M; and the concentration
of a PKC inhibitor is 50 nM to 100 .mu.M.
[0069] In the present invention, although obvious to a person
skilled in the art, it is preferable that culturing is performed in
a low-oxygen condition (oxygen concentration: 5%) as the culture
conditions for using a naive medium.
[0070] The word "transient" in the step (iii) of the present
invention means about five days or more, or about ten days or more,
and preferably about 10 to 20 days, and particularly preferably
about 10 to 14 days.
[0071] In the present invention, the word "allowing expression of
the gene introduced in the step (i)" in the step (iii) means, for
example, an action of inducing expression of the introduced gene by
addition of doxycycline.
[0072] In the present invention, examples of the method for
determining whether the produced naive pluripotent stem cells is in
a naive state or not include a method of determining an expression
amount of naive markers of the cells by quantitative PCR, although
obvious to a person skilled in the art. Herein, the examples of the
naive marker include DPPA3, ESRRB, TFCP2L1, KLF4, KLF5, and TBX3.
Alternatively, the determination can be carried out depending on
morphologies, because the primed pluripotent stem cells form mainly
a single-layered flat colony, while the naive pluripotent stem
cells form mainly double-layered (dome-shaped) in the colony
morphology.
[0073] In the present invention, examples the method for
determining pluripotency of the produced naive pluripotent stem
cells include a method for determining an expression amount of the
pluripotent marker of the cells by quantitative PCR, although
obvious to a person skilled in the art. Herein, examples the
pluripotent marker include OCT3/4, Nanog, Sox2, SSEA4, and the
like. Alternative determination methods include an embryoid body
(EB) method, and a teratoma formation method (teratoma).
[0074] In the maintaining method of the naive pluripotent stem
cells in the present invention, since induction of expression of an
introduced gene is not necessarily needed, it may be or may not be
carried out. Preferably, the induction of expression of the
introduced gene is not carried out and culturing is performed in a
naive medium in which various compounds are added.
[0075] In the present invention, the produced naive pluripotent
stem cells maintain an undifferentiated state, in other words,
highly express ESRRB that is one of the naive markers, and has very
high pluripotency. Accordingly, they can be used for (1) induction
of differentiation into desired various cells, (2) screening of
candidate compounds for pharmaceutical agents using the
differentiation-induced cells; (3) generation of tissue for
regeneration medicine from the differentiation-induced cells, (4)
transplantation of the generated tissue into a patient; (5) an
organ regeneration method by transplantation of naive-state iPS
cells into blastocysts, and the like.
[0076] In the present invention, cells capable of induction of
differentiation of the naive pluripotent stem cells are not
particularly limited, and examples thereof include cardiomyotic
cell, a nerve cell, insulin-production cell, glomerulus endothelial
cell, mesangium cell, Bowman's epithelial cell, and blood vessel
endothelial cell.
[0077] In the present invention, a method for
differentiation-inducing the naive pluripotent stem cells is not
particularly limited, and examples of a method for
differentiation-inducing into the nerve cell include a SDIA
(Stromal cell-Derived Inducing Activity) method (Proceedings of the
National Academy of Sciences of the United States of America, Vol.
99, No. 3, 1580-1585, 2002).
[0078] In the present invention, cells that have been
differentiation-induced using naive pluripotent stem cells can be
used for screening drug candidate compounds for various diseases.
For example, by adding the drug candidate compounds singly or in
combination with other drugs into the differentiation-induced
cells, the morphology or functional change of the cells, increase
and decrease of various factors, gene expression profiling, and the
like, are detected so as to carry out evaluation. Herein, the cells
are preferably cells having the same phenotype as that of disease
to be treated, and more preferably cells differentiation-induced
from the naive pluripotent stem cells produced from somatic cells
derived from a patient having a disease.
[0079] In the present invention, tissue can be generated from
differentiation-induced cells using naive pluripotent stem cells,
and the generated tissue can be used in the field of the
regeneration medicine. For example, damaged nerve tissue can be
normalized by replacing the damaged nerve tissue by normal tissue
derived from nerve tissue of present invention. This makes it
possible to cure diseases derived from damaged nerve cells.
Examples of such diseases include Parkinson's disease, Alzheimer's
disease, retinal pigmentary degeneration, amyotrophic lateral
sclerosis, optic neuromyelitis, optic neuritis, acute disseminated
encephalomyelitis, allergic encephalomyelitis, spinal cord damage,
transverse myelitis, spinocerebellar degeneration, chronic
inflammatory demyelinating encephalopathy (CIDP), Guillain-Barre
syndrome, multiple sclerosis, epilepsy, Parkinson's syndrome, Down
syndrome, schizophrenic disorder, neurodystonia, Huntington's
disease, age-related macular degeneration, and inner ear
deafness.
[0080] In the present invention, as a transplanting method of the
generated tissue into a patient, although obvious to a person
skilled in the art, for example, when nerve cells are transplanted,
transplantation can be carried out according to the method
described in Nature Neuroscience, Vol. 2, No. 12, page 1137-1140,
1999.
EXAMPLES
[0081] Hereinafter, the present invention is described in detail
with reference to Examples mentioned below, but the present
invention is not limited thereto.
[0082] Biological Experiment Examples are described below. The
advantageous effect of the compound of the present invention was
verified based on these experiment methods.
Example 1
[0083] Making iPS Cells Naive
1.1 Culturing of primed human iPS cells
[0084] As human iPS cells, WD39 cells (Imaizumi et al. Molecular
Brain 2012, 5:35) and 201B7 cells (RIKEN BioResource Center) widely
used as a control line were used.
[0085] The human iPS cells were cultured using a medium in the
primed conditions shown in Table 1 using Mitomycin C-treated or
radiation-treated mouse embryonic fibroblast line (STO cells) or
mouse embryonic fibroblasts (MEF cells) as feeder cells.
Subculturing was performed once every five to seven days. In
subculturing, colonies were detached by using a dissociation
solution (0.25% Trypsin, 1 mg/ml Collagenase IV, 20% KSR, and 1 mM
CaCl2)/PBS), finely crushed by pipetting, and then seeded. The iPS
cells seeded in a 10-cm dish were subjected to a gene introduction
test.
1.2 Generation of Gene-Introduced Cell Line
[0086] The medium of the iPS cells (10-cm dish) cultured in the
conditions of "1.1 Culturing of primed human iPS cells" mentioned
above was removed and washed with 7 mL of PBS once, and 1 mL of
dissociation solution was then added thereto. After 1 to 2 minutes,
the dissociation solution was removed, and 7 mL of PBS was added.
The dish was slightly shaken and only the feeder cells were
detached, and then PBS was removed. Furthermore, this was washed
with PBS once, and 3 mL of the medium of the primed conditions
shown in Table 1 was added to the dish, and colonies of the iPS
cells were detached using a scraper and then collected into a 15-mL
tube. The iPS cells in an amount of 200 g were centrifuged for 5
minutes to remove a supernatant, 1 mL of TrypLE.TM. Select (Life
Technologies) was added and reacted at 37.degree. C. for 5 minutes.
To the reacted product, 2 mL of Trypsin Inhibitor (Life
Technologies) was added, followed by pipetting 10 to 20 times using
P1000 PIPETMAN so as to dissociate single cells. The volume was
increased by addition of 7 mL of medium, and the resulting product
was allowed to pass through 70-.mu.m Cell Strainer. The iPS cells
as single cells were used for gene introduction.
[0087] In the gene introduction, three types of vectors: a reverse
tetracycline-controlled transactivator (rtTA) expression vector
(rtTA: rtTA-Advanced sequence (Clontech) was used), a PiggyBac
Transposase expression vector (see, Proc Natl Acad Sci USA. 2011
Jan. 25; 108(4): 1531-6), a vector which doxycycline-dependently
expresses Yamanaka four factors, KLF2 and NANOG, and TOMATO as a
fluorescence protein (OKSM, KLF2, NANOG, and TOMATO) were
introduced (FIG. 1) using Gene juice (registered trademark)
Transfection Reagent (Novagen), and cultured in the same manner as
in the above-mentioned "1.1 Culturing of primed human iPS cells."
Five to ten days after the gene introduction, doxycycline was added
and observed under a fluorescence microscope after 8 to 12 hours
had passed. A colony in which fluorescence of TOMATO was observed
was determined to be a cell line in which a doxycycline-dependent
expression induction system functions, and the colony was picked up
and cloned. Later than that, doxycycline was not added, and
culturing was performed in the same manner as in the
above-mentioned "1.1 Culturing of primed human iPS cells."
1.3 Making iPS Cells Naive
[0088] The iPS cells gene-introduced and cloned in "1.2 Generation
of gene-introduced cell line" were dissociated into single cells by
using a dissociation solution and TrypLE.TM. Select, and seeded in
a 6-well plate at 1.times.10.sup.5 cells/well in which MEF cells
had been seeded as feeder. At this time, as a medium, 2iLFA+Go6983
medium shown in Table 1 (hereinafter, also abbreviated as "the
medium of the present invention") was used, and cultured in low
oxygen in a state in which doxycycline (1 .mu.g/mL) was further
added so as to induce expression of the introduced gene (Passage 1;
P1). Note here that on the first day of the start of making naive,
Y27632 (ROCK inhibitor) was added so as to be 10 .mu.M, resulting
in undergoing cell death. On day 5 to 7 after the start of making
naive, subculturing was performed. The medium was removed, and then
350 .mu.L of 0.25% Trypsin-EDTA was added in each well to cause
reaction for one minute at 37.degree. C. The reaction was stopped
by Trypsin Inhibitor, and then 5 mL of medium was added thereto.
Pipetting was carried out several times using PIPETTE AID to detach
colonies of iPS cells from a sheet of feeder cells. The colonies
were collected in a 15-mL tube. After centrifugation, a supernatant
was removed, and 200 .mu.L of medium was added, followed by
pipetting using P200 PIPETMAN about 40 times to be dissociated into
single cells. All amount of cells were seeded in a new 6-well plate
in which MEF cells were cultured as the feeder cells (Passage 2).
Also after subculturing, similar to P1, culturing was performed in
the condition in which doxycycline was added into the medium of the
present invention. Five to seven days after the subculturing,
colony morphology of the iPS cells was observed under microscope,
and second subculturing was performed. After the second
subculturing or later, doxycycline was not added. Five to seven
days after the second subculturing, colony morphology of the iPS
cells was observed under microscope, and third subculturing was
performed. The fourth subculturing or later was performed in the
same manner. Note here that at the time of the second and third
subculturing, a part of cells at Passage 2 and Passage 3 were
collected for qPCR analysis (see FIG. 8).
Example 2
[0089] Analysis of Gene Expression Profile by Making Naive
[0090] In this experiment, 201B7 cells were used as the iPS
cells.
[0091] Cells used for analysis were 201B7 cells cultured in the
medium in the primed condition (see "1.1 Culturing of primed human
iPS cells"), and 201B7 cells at passage 2 and Passage 3 generated
in "1.3 Making iPS cells naive." RNAs were collected from the
respective cells using RNeasy mini kit (QIAGEN), subjected to
reverse transcription using ReverTra Ace (registered trademark),
and then subjected to qPCR using SYBR (registered trademark) Premix
Ex TaqII (clontech). The primer sets are shown in Table 2.
[0092] The results are shown in FIG. 2. Expressions of NANOG and
OCT3/4 as the pluripotent markers were maintained even a state
becomes naive. Furthermore, in genes such as KLF4, DPPA3, ESRRB,
TFCP2L1, KLF5 and TBX3 exhibiting high expression in mouse ES cells
as naive pluripotent stem cells, expression was increased by making
naive. High expression was maintained as compared with in the
primed state also after addition of doxycycline was stopped
(Passage 3). Furthermore, expression of Lefty as a primed gene
marker was reduced by making naive. From the above-mentioned
results, it was found that when the method of the present invention
was used, the iPS cells were transitioned to the naive state at
least after passage 2. Furthermore, it was found that after the iPS
cells were transitioned to the naive state, in order to maintain
the naive state, induction of expression of the introduced gene was
not needed, and that only the medium of the present invention was
sufficient.
Example 3
[0093] Effect of Addition Timing of Go6983 on Making Naive
[0094] In this experiment, an effect of timing of addition of
Go6983 on making naive was considered. A 201B7 cell culture medium
at passage 1 in "1.3 Making iPS cells naive" (a medium of
2iLFA+Go6983 in Table 1) was changed to a medium of KSR 2iLFA of
Table 1 or a medium of KSR 2iLFA+Go6983 of Table 1, expression of
the introduced gene was induced by addition of doxycycline,
followed by culturing in low oxygen. The passage 1 cultured in each
condition was collected 5 to 7 days after the subculturing, and
subjected to qPCR analysis. Results are shown in FIG. 3. As is
apparent from FIG. 3, the iPS cells cultured in a medium into which
Go6983 had been added had higher expression of ESRRB. From the
results, it was found that the addition of Go6983 at the time of
induction of expression of the introduced genes by doxycycline
promoted the naive state of the iPS cells.
Example 4
[0095] Effect of Addition of Forskolin and A83-01 on Making
Naive
[0096] An ESRRB gene is an important gene that controls
self-replication in mouse ES cells (see, Cell Stem Cell, 11,491-504
(2012)). In the present invention, as is apparent from the results
of Example 1, regardless of induction of expression of introduced
genes, high expression of ESRRB was maintained. On the contrary, in
a reset cell described in Non-Patent Literature 3, expression of
ESRRB is hardly found (see Non-Patent Literature 3). One of the
differences between the condition of the present invention and the
condition described in Non-Patent Literature 3 is that in the
present invention, Forskolin and A83-01 were added into the medium
condition described in Non-Patent Literature 3. Then, the effect of
this difference on making naive (expression of ESRRB and colony
morphology) was considered.
[0097] In this experiment, a method for making naive was carried
out in the same manner as in the "1.3 Making iPS cells naive"
except that the medium of the present invention (a medium of
2iLFA+Go6983 of Table 1) was used as a medium for considering
making naive, and the medium of 2iL+Go6983 of Table 1 was used as a
comparative medium. Evaluation whether or not the cells were made
to be a naive state carried out using ESRRB gene expression and the
colony morphology of 201B7 cells at passage 2 and Passage 3
cultured in respective media as indicators.
[0098] Results are shown in FIG. 4. In both media, during addition
of doxycycline, the expression of ESRRB was increased, and a colony
morphology was dome-shaped similar to the naive mouse ES cells.
However, in the iPS cells cultured in a 2iL+Go6983 medium, when the
addition of doxycycline was stopped, the expression of ESRRB was
remarkably reduced (about 10% of the expression at the time of
addition of doxycycline), and flat colonies, which are
characteristics of the primed type, were increased. On the
contrary, in the iPS cells cultured in the medium of the present
invention (2iLFA+Go6983 medium in Table 1), even after the addition
of doxycycline was stopped, the expression of ESRRB was reduced,
but the reduction was more slightly as compared with the reduction
in 2iL+Go6983 conditions (about 50% of the expression at the time
of addition of doxycycline), and the dome-shaped colony morphology
was maintained.
[0099] From the results, it was found that the addition of
Forskolin and A83-01 was needed in order to maintain the naive
state of the iPS cells even after stopping the induction of
expression of the introduced gene.
[0100] Note here that in the Non-Patent Literature 3, Go6983 was
added at the timing when doxycycline was removed. However, since
this test was focused on the effect of Forskolin and A83-01, Go6983
was added at the time when doxycycline was added as in the method
of the present invention. As is apparent from Example 2, expression
of ESRRB was higher when Go6983 was added at the time of
doxycycline. Therefore, the expression of ESRRB in the reset cell
in Non-Patent Literature 3 was further lower than the results of
this experiment, its expression was hardly found as is apparent
from Non-Patent Literature 3, transcription factor network in the
reset cell was weak (see Non-Patent Literature 3). On the contrary,
in the naive iPS cells produced and maintained by the method of the
present invention, expression of the transcription factor that is
the same as in the naive mouse ES cells is found, it is considered
that the same transcription factor network as in the naive mouse ES
cells is formed. Therefore, it is shown that the naive iPS cells
produced and maintained by the method of the present invention are
in the advanced naive state as compared with the reset cells
described in Non-Patent Literature 3.
Example 5
[0101] Comparison of Reprogramming Efficiency by Combination of
Reprogramming Factors
[0102] One of the differences between the condition of the present
invention and the condition described in Non-Patent Literature 3 is
that in the present invention, the Yamanaka four factors were added
to the reprogramming factors described in Non-Patent Literature 3
(KLF2 and NANOG). Thus, the effect of this difference on making
naive (expression of ESRRB) was considered.
[0103] The WD39 cells cultured in the conditions of the
above-mentioned "1.1 Culturing of primed human iPS cells" were
subjected to gene introduction using a technique similar to that in
"1.2 Generation of gene-introduced cell line." In the gene
introduction, three types of vectors: a reverse
tetracycline-controlled transactivator (rtTA) expression vector
(rtTA: rtTA-Advanced sequence (Clontech) was used), a PiggyBac
Transposon expression vector (see Proc Natl Acad Sci USA. 2011 Jan.
25; 108(4): 1531-6), and a vector which doxycycline-dependently
expresses a reprogramming factor and fluorescence protein were
introduced (FIG. 9), and cultured in the same manner as in the
above-mentioned "1.1 Culturing of primed human iPS cells." Note
here that as the combination of the reprogramming factor and the
fluorescence protein, three types: (1) KLF2, NANOG and Venus, (2)
Yamanaka four factors and Cerulean, (3) Yamanaka four factors,
KLF2, NANOG and TOMATO, were used. The next day to four days after
the gene introduction, doxycycline was added. From the next day of
the addition of doxycycline, selection by drug was carried out. As
the selection, neomycin (100 .mu.g/mL) or puromycin (1 .mu.g/mL)
was added depending on the types of doxycycline-dependently
expressed resistance genes. Five days after addition of
doxycycline, cells were collected and subjected to gene expression
analysis by qPCR. As a result, in the cells in which the genes of
(3) Yamanaka four factors, KLF2, NANOG and TOMATO had been
introduced, expression of ESRRB was synergistically higher than the
cells in which (1) KLF2, NANOG and Venus, (2) Yamanaka four factors
and Cerulean had been introduced (FIG. 5). Consequently, naive iPS
cells produced by introducing six genes of Yamanaka four factors,
KLF2, and NANOG were found to be in a state in which the naive
state was advanced as compared with the reset cells described in
Non-Patent Literature 3.
[0104] From the above-mentioned results of Examples 3 to 5, the
followings were clarified.
[0105] (1) Addition of a PKC inhibitor (for example, Go6983) at the
time of inducing expression of an introduced gene promotes making
naive.
[0106] (2) Introduction of six genes including Yamanaka four
factors promotes making naive as compared with the case of
introduction of only two genes of KLF2 and NANOG.
[0107] (3) Addition of a cAMP production promoter (for example,
Forskolin) and a TGF-.beta. inhibitor (for example, A83-01) allows
the naive state of cells to be maintained.
[0108] As mentioned above, naive pluripotent stem cells (for
example, human iPS cells) produced and maintained by the method
according to the present invention are in a state in which a naive
state is much further advanced as compared with the reset cells
described in Non-Patent Literature 3, and they sufficiently
function as the naive pluripotent stem cells.
Example 6
[0109] Neural Differentiation Induction from iPS Cells that were
Made Naive
[0110] Neural differentiation induction of primed iPS cells and iPS
cells that were made naive were carried out by an SDIA method. The
SDIA method is a method for inducing neural differentiation by
culturing iPS/ES cells using a mouse stromal cell line (PA6 cells,
RIKEN BioResource Center) as feeder cells (see Kawasaki et al.
Neuron. 2000 October; 28(1): 31-40). For usual culturing of PA6
cells, .alpha.MEM+10% FBS was used. When the SDIA method was
carried out, the medium was changed to the SDIA medium of Table 1.
When the SDIA method was carried out from the primed state,
colonies were made into single cells by the same method as in the
above-mentioned "1.2 Generation of gene-introduced cell line" but,
for the purpose of suppressing the cell death, from one hour before
the start of the experiment, Y27632 as a ROCK inhibitor was added
so as to be 10 .mu.M. After cells were seeded on the PA6 cells,
Y27632 was not added. The cells were seeded to a 12-well plate so
that the number of the cells was 2.times.10.sup.3 to
2.times.10.sup.4 cells/well. When the SDIA method was carried out
from the naive state, cells at passage 3 or Passage 4, which had
been generated in the above-mentioned "1.3 Making iPS cells naive,"
were made into colonies of single cells by a method using
Trypsin-EDTA. Note here that for the purpose of suppressing the
cell death, from one hour before the start of experiment, Y27632 as
the ROCK inhibitor was added so as to be 10 .mu.M. After the cells
were seeded on the PA6 cells, Y27632 was not added. The cells were
seeded to a 12-well plate so that the number of the cells was
2.times.10.sup.3 to 2.times.10.sup.4 cells/well. The cells were
fixed by 4% PFA on day 10 of the SDIA method, and MAP2 (antibody
M4403, sigma) was subjected to immunohistostaining. Each colony was
observed under fluorescence microscope to determine whether or not
induction of the MAP2-positive nerve was induced. The results are
shown in FIG. 6.
[0111] In the iPS cells were made naive, the rate of colonies in
which MAP2-positive cells were present was remarkably higher than
that of the primed iPS cells. This result was observed in both
201B7 cells and WD39 cells used. This result showed that the iPS
cells that were made naive by the method of the present invention
had excellent differentiation potency.
Example 7
[0112] Induction of Differentiation of Astrocyte from iPS Cells
that were Made Naive
[0113] Primed iPS cells and iPS cells that were made naive were
substituted to neural differentiation induction by a method via
Neurosphere (see Stem Cells. 2008 December; 26(12): 3086-98). When
astrocyte differentiation from the primed state was carried out, a
colony in a shape of aggregates was detached using a dissociation
solution, and floating-culturing was carried out in a medium in
which bFGF had been removed from a medium in the primed condition
in the Table 1 so as to form an embryoid body. When the astrocyte
differentiation was carried out from the naive state, pipetting was
repeated strongly to detach a colony in a shape of aggregates, and
culturing was performed in an N2B27 medium (a basal medium of the
naive medium) to form an embryoid body. Note here that the same
operation was carried out in the primed iPS cells and iPS cells
that were made naive except for a method of detaching the colony at
the time of formation of the embryoid body and a medium.
Furthermore, during formation of the embryoid body, in order to
promote differentiation into the nervous system, LDN193189 was
added so as to be 100 nM. Seven days after the formation of the
embryoid body, the embryoid body was collected and 1 mL of
TrypLE.TM. Select was added thereto, and the mixture was reacted at
37.degree. C. for 10 minutes. After 2 mL of Trypsin Inhibitor was
added, pipetting was carried out using P1000 PIPETMAN 20 to 30
times so as to be dissociated into single cells. The dissociated
cells were adjusted to be 2.times.10.sup.3 cells/ml, and
floating-culturing was performed in an MHM medium in which 2% B27
and 20 ng/ml bFGF were added so as to form primary Neurosphere.
Seven days after the formation of the primary Neurosphere,
Neurosphere was collected, 1 mL of TrypLE.TM. Select was added
thereto, and the mixture was reacted at 37.degree. C. for 10
minutes. After 2 mL of Trypsin Inhibitor was added, pipetting was
carried out using P1000 PIPETMAN 20 to 30 times so as to be
dissociated into single cells. The dissociated cells were adjusted
to be 2.times.10.sup.3 cells/ml, and floating-culturing was
performed in an MHM medium in which 2% B27 and 20 ng/ml bFGF were
added to form secondary Neurosphere. Seven days after the formation
of the secondary Neurosphere, the secondary Neurosphere was seeded
onto a cover glass coated with poly-L-ornithine and fibronectin so
as to promote differentiation. As a medium for differentiation, an
MHM medium containing 2% B27, 2% FBS and 20 ng/ml hLIF was used.
Ten days after seeding on the cover glass, the cells were fixed by
4% PFA, immunohistostaining was carried out with respect to
.beta.III-Tubulin as a nerve marker and GFAP as an astrocyte marker
(antibody T8660, sigma, and antibody 2.2B10, Thermo Fisher
Scientific).
[0114] Results are shown in FIG. 7. In the iPS cells that were made
naive, the rate of differentiation into astrocyte was remarkably
higher as compared with that of the primed iPS cells. This result
was observed in both the 201B7 cells (B7 in the drawing) and WD39
cells (WD in the drawing). The result showed that the iPS cells
that were made naive by the method of the present invention had
excellent differentiation potency.
TABLE-US-00001 TABLE 1 Small molecule, Culture Medium Name Medium
etc. condition Primed DMEM/F12 4 ng/mL bFGF 37.degree. C. condition
20% KSR 5% CO.sub.2 1% NEAA 1 mM L-glutamine 0.1 mM
-2-mercaptoethanol Penicillin/ streptomycin 2iLFA + Go6983 DMEM/F12
and 10 ng/mL hLIF 37.degree. C. Neurobasal(1:1) 1 .mu.M CHIR99021
5% CO.sub.2 1% N2 1 .mu.M PD0325901 5% O.sub.2 2% B27 5 .mu.M
Go6983 1% NEAA 10 .mu.M Forskolin 1 mM L-glutamine 0.5 .mu.M
A-83-01 50 .mu.g/mL BSA 0.1 mM 2-mercaptoethanol Penicillin/
streptomycin 2iL + Go6983 DMEM/F12 and 10 ng/mL hLIF 37.degree. C.
Neurobasal (1:1) 1 .mu.M CHIR99021 5% CO.sub.2 1% N2 1 .mu.M
PD0325901 5% O.sub.2 2% B27 5 .mu.M Go6983 1% NEAA 1 mM L-glutamine
50 .mu.g/mL BSA 0.1 mM 2-mercaptoethanol Penicillin/ streptomycin
KSR 2iLFA KO-D-MEM 10 ng/mL hLIF 37.degree. C. 20% KSR 3 .mu.M
CHIR99021 5% CO.sub.2 1% NEAA 10 .mu.M Forskolin 5% O.sub.2 1 mM
L-glutamine 1 .mu.M PD0325901 0.2 mM 0.5 .mu.M A-83-01
2-mercaptoethanol Penicillin/ streptomycin KSR KO-D-MEM 10 ng/mL
hLIF 37.degree. C. 2iLFA + Go6983 20% KSR 3 .mu.M CHIR99021 5%
CO.sub.2 1% NEAA 10 .mu.M Forskolin 5% O.sub.2 1 mM L-glutamine 1
.mu.M PD0325901 0.2 mM 0.5 .mu.M A-83-01 2-mercaptoethanol 5 .mu.M
Go6983 Penicillin/ streptomycin SDIA medium G-MEM 37.degree. C. 10%
KSR 5% CO.sub.2 1 mM Pyruvate 2 mM L-glutamine 0.1 mM
2-mercaptoethanol
TABLE-US-00002 TABLE 2 Gene Forward primer Reverse primer
Endo-NANOG gacactggctgaatcct accctccatgagattga tcctct ctggat (SEQ
ID No: 1) (SEQ ID No: 2) Endo-OCT3/4 agtttgtgccagggttt
acttcaccttccctcca ttg acc (SEQ ID No: 3) (SEQ ID No: 4) Endo-KLF4
gccagaaagcactacaa ttggcattttgtaagtc tcatgg caggaa (SEQ ID No: 5)
(SEQ ID No: 6) Lefty agctgcacaccctggac gtcattggtgcttcagg ctt gtca
(SEQ ID No: 7) (SEQ ID No: 8) DPPA3 Takara perfect real time
(HA216940) ESRRB Takara perfect real time (HA095808) TFCP2L1 Takara
perfect real time (HA193431) KLF5 Takara perfect real time
(HA224420) TBX3 Takara perfect real time (HA142445)
INDUSTRIAL APPLICABILITY
[0115] The present invention makes it possible to stably produce
pluripotent stem cells maintaining characteristics of the naive
state and having pluripotency, and therefore, can be practically
applied to clinical use and drug evaluation, and the like.
Sequence Listing Free Text
[0116] SEQ ID NO 1: primer SEQ ID NO 2: primer SEQ ID NO 3: primer
SEQ ID NO 4: primer SEQ ID NO 5: primer SEQ ID NO 6: primer SEQ ID
NO 7: primer SEQ ID NO 8: primer
Sequence CWU 1
1
8123DNAArtificialForward Primer 1gacactggct gaatccttcc tct
23223DNAArtificialReverse Primer 2accctccatg agattgactg gat
23320DNAArtificialForward Primer 3agtttgtgcc agggtttttg
20420DNAArtificialReverse Primer 4acttcacctt ccctccaacc
20523DNAArtificialForward Primer 5gccagaaagc actacaatca tgg
23623DNAArtificialReverse Primer 6ttggcatttt gtaagtccag gaa
23720DNAArtificialForward Primer 7agctgcacac cctggacctt
20821DNAArtificialReverse Primer 8gtcattggtg cttcagggtc a 21
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