U.S. patent application number 12/292717 was filed with the patent office on 2010-03-25 for efficient method for nuclear reprogramming.
This patent application is currently assigned to Kyoto University. Invention is credited to Michiyo Koyanagi, Shinya Yamanaka.
Application Number | 20100075421 12/292717 |
Document ID | / |
Family ID | 40755366 |
Filed Date | 2010-03-25 |
United States Patent
Application |
20100075421 |
Kind Code |
A1 |
Yamanaka; Shinya ; et
al. |
March 25, 2010 |
Efficient method for nuclear reprogramming
Abstract
A method of preparing induced pluripotent stem cells, comprising
a nuclear reprogramming step with a nuclear reprogramming factor in
the presence of miRNA, wherein said miRNA has a property of
providing a higher nuclear reprogramming efficiency in the presence
of said miRNA than in the absence thereof.
Inventors: |
Yamanaka; Shinya; (Kyoto,
JP) ; Koyanagi; Michiyo; (Kyoto, JP) |
Correspondence
Address: |
GREENBLUM & BERNSTEIN, P.L.C.
1950 ROLAND CLARKE PLACE
RESTON
VA
20191
US
|
Assignee: |
Kyoto University
Kyoto
JP
|
Family ID: |
40755366 |
Appl. No.: |
12/292717 |
Filed: |
November 25, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/JP2008/059586 |
May 23, 2008 |
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12292717 |
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60996893 |
Dec 10, 2007 |
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60996893 |
Dec 10, 2007 |
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Current U.S.
Class: |
435/455 ;
435/366 |
Current CPC
Class: |
C12N 5/0696 20130101;
C12N 2501/604 20130101; C12N 2501/65 20130101; C12N 2501/606
20130101; C12N 15/113 20130101; C12N 2501/602 20130101; C12N
2501/608 20130101; C12N 2330/10 20130101; C12N 2501/603 20130101;
C12N 2501/605 20130101; C12N 2310/141 20130101; C12N 2510/00
20130101 |
Class at
Publication: |
435/455 ;
435/366 |
International
Class: |
C12N 15/09 20060101
C12N015/09; C12N 5/071 20100101 C12N005/071 |
Claims
1. A method of preparing induced pluripotent stem cells, comprising
nuclear reprogramming at least one somatic cell with nuclear
reprogramming factor and at least one miRNA, wherein the at least
one miRNA increases efficiency of the nuclear reprogramming of the
at least one somatic cell compared to nuclear reprogramming of the
at least one somatic cell with the nuclear reprogramming factor in
the absence of the at least one miRNA.
2. The method according to claim 1, wherein the at least one miRNA
is expressed in embryonic stem cells at a higher level than in
somatic cells.
3. The method according to claim 1, wherein a gene encoding the
nuclear reprogramming factor and/or the at least one miRNA is
introduced into the at least one somatic cell.
4. The method according to claim 3, wherein a vector comprising the
gene and/or a vector encoding the at least one miRNA is introduced
into the at least one somatic cell.
5. The method according to claim 4, wherein the vector comprising
the gene or encoding the at least one miRNA is a retroviral
vector.
6. The method according to claim 3, wherein the gene is selected
from an Oct family gene, a Klf family gene, and a Sox family
gene.
7. The method according to claim 3, wherein the gene is selected
from Oct3/4, Klf4, and Sox2.
8. The method according to claim 3, wherein the nuclear
reprogramming factor comprises Oct3/4, Klf4, and Sox2.
9. The method according to claim 3, wherein the at least one miRNA
is introduced into the at least one somatic cell as primary
miRNA.
10. The method according to claim 3, wherein the at least one miRNA
is introduced into the at least one somatic cell as pre-miRNA.
11. The method according to claim 1, wherein the at least one miRNA
comprises at least one miRNA represented by SEQ ID NOs: 1 to
14.
12. The method according to claim 1, wherein the at least one miRNA
comprises at least one miRNA contained in miRNA cluster
hsa-miR-302-367 cluster, hsa-miR-371-373 cluster and
hsa-miR-520c.
13. The method according to claim 1, wherein the at least one miRNA
regulates DNA methylation.
14. The method according to claim 11, wherein the at least one
miRNA regulates de novo DNA methylation.
15. The method according to claim 11, wherein the at least one
miRNA down-regulates DNA methylation.
16. The method according to claim 1, wherein the at least one miRNA
comprises at least 10 contiguous nucleotides of at least one miRNA
represented by SEQ ID NOs: 1 to 14.
17. The method according to claim 1, wherein the at least one miRNA
comprises at least 30 contiguous nucleotides of at least one miRNA
represented by SEQ ID NOs: 1 to 14.
18. The method according to claim 1, wherein the at least one miRNA
comprises at least 60 contiguous nucleotides of at least one miRNA
represented by SEQ ID NOs: 1 to 14.
19. The method according to claim 1, wherein the nuclear
reprogramming factor does not include c-Myc and/or Sox2.
20. The method according to claim 1, wherein the at least one miRNA
comprises hsa-miR-302-367 cluster, hsa-miR-371-373 cluster and
hsa-miR-520C miRNA.
21. The method according to claim 20, wherein the nuclear
reprogramming factor comprises an Oct family gene member, a Sox
family gene member, and a Klf family gene member.
22. The method according to claim 1, wherein the at least one miRNA
comprises mmu-miR-295/295* and 294/294*.
23. The method according to claim 1, wherein the at least one miRNA
comprises hsa-miR-302-367 cluster and hsa-miR-371-373 cluster
miRNA.
24. The method according to claim 1, wherein the nuclear
reprogramming factor comprises a Klf family gene, and an Oct family
gene.
25. The method according to claim 24, wherein the nuclear
reprogramming factor further comprises a Myc family gene.
26. The method according to claim 24, wherein the nuclear
reprogramming factor further comprises a Sox family gene.
27. The method according to claim 25, wherein the nuclear
reprogramming factor further comprises a Sox family gene.
28. The method according to claim 24, wherein the nuclear
reprogramming factor comprises KLF4 and OCT3/4.
29. A method of increasing the efficiency of nuclear reprogramming
comprising: adding a nuclear reprogramming factor and at least one
miRNA to at least one somatic cell so that the number of induced
pluripotent stem cells produced is greater than in the absence of
the added miRNA.
30. An induced pluripotent stem cell induced by reprogramming a
somatic cell, wherein the reprogramming is performed by adding at
least one miRNA and in the absence of eggs, embryos, or embryonic
stem (ES) cells.
31. The induced pluripotent stem cell according to claim 30,
wherein the induced pluripotent stem cell is a human cell.
32. An induced pluripotent stem cell obtained by the method of
claim 1.
33. An induced pluripotent stem cell obtained by the method of
claim 29.
34. A somatic cell derived by inducing differentiation of the
pluripotent stem cell according to claim 32.
35. A somatic cell derived by inducing differentiation of the
pluripotent stem cell of claim 33.
36. The method according to claim 25, wherein the nuclear
reprogramming factor excludes a Sox family gene.
37. The method according to claim 26, wherein the nuclear
reprogramming factor excludes a Myc family gene.
38. The method according to claim 1, where the at least one somatic
cell comprises a plurality of somatic cells.
Description
PRIOR RELATED APPLICATIONS
[0001] This application is a continuation-in-part of
PCT/JP2008/59586, filed May 23, 2008, which claims priority of U.S.
Provisional Application No. 60/996,893, filed Dec. 10, 2007, and
this application also claims priority of U.S. Provisional
Application No. 60/996,893, filed Dec. 10, 2007. The entire
disclosures of each of the above-cited applications are considered
as being part of this application and are expressly incorporated by
reference herein in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to efficient methods for
preparing induced pluripotent stem cells through reprogramming of
somatic cells, to induced pluripotent stem cells, to uses of
induced pluripotent stem cells and to somatic cells derived by
inducing differentiation of said pluripotent cells. The present
invention also relates to nuclear reprogramming factors and to
miRNAs involved in generating induced pluripotent stem cells. The
present invention also relates to screening methods, treatments,
and therapies involving the use of induced pluripotent stem
cells.
BACKGROUND OF THE INVENTION
[0003] Embryonic stem cells (ES cells) are stem cells established
from human or mouse early embryos which have a characteristic
feature that they can be cultured over a long period of time while
maintaining pluripotent ability to differentiate into all kinds of
cells existing in living bodies. Human embryonic stem cells are
expected for use as resources for cell transplantation therapies
for various diseases such as Parkinson's disease, juvenile
diabetes, and leukemia, taking advantage of the aforementioned
properties. However, transplantation of ES cells has a problem of
causing rejection in the same manner as organ transplantation.
Moreover, from an ethical viewpoint, there are many dissenting
opinions against the use of ES cells which are established by
destroying human embryos.
[0004] If dedifferentiation of patients' own differentiated somatic
cells could be induced to establish cells having pluripotency and
growth ability similar to those of ES cells (these cells are
referred herein to as "induced pluripotent stem cells" or "iPS
cells," though they are sometimes called "embryonic stem cell-like
cells" or "ES-like cells"), it is expected that such cells would be
useful as ideal pluripotent cells, free from rejection or ethical
difficulties. Recently, it has been reported that such iPS cells
can be produced from differentiated cells of mouse or human, which
has created a great sensation (International Publication No.
WO2007/069666 A1; Takahashi et al., Cell 126:663-76, 2006;
Takahashi et al., Cell 131:861-72, 2007; Yu et al., Science
318:1917-20, 2007; and Park et al., Nature 451:141-46, 2008, herein
incorporated by reference in their entireties). Thus, the term
"induced pluripotent stem cells (iPS cells)" refers to cells having
similar properties to those of ES cells, and more specifically the
term includes undifferentiated cells which are reprogrammed from
somatic cells and have pluripotency and proliferation potency.
However, this term is not to be construed as limiting in any sense,
and should be construed to have its broadest meaning.
[0005] These methods include a reprogramming step through
introduction of a plurality of specific factors (for example, four
factors of Oct3/4, Sox2, Klf4, and c-Myc can be used in Takahashi
et al., Cell 126:663-76, 2006), and the introduction of these
factors is mediated by viral vectors such as retroviral or
lentiviral vectors. However, all previously reported nuclear
reprogramming methods mediated by the introduction of genes involve
a problem of low efficiency in which only a small number of induced
pluripotent stem cells can be obtained. In particular, there is a
problem in that, if reprogramming is carried out in somatic cells
through the introduction of three factors (namely, Oct3/4, Sox2,
and Klf4) excluding c-Myc, then the production efficiency of
induced pluripotent stem cells becomes low. Nevertheless, the
efficient production of iPS cells without the use of c-Myc would
provide certain advantages, as c-Myc is suspected to cause
tumorigenesis in tissues and in chimeric mice generated from
induced pluripotent stem cells.
[0006] It is known that various small RNAs are expressed in cells.
Examples of small RNA include RNA molecules of about 18-25
nucleotides in length which can be cleaved out with a dicer, an
RNase specific to double-stranded RNA. Small RNA is mainly
classified into siRNA (small interfering RNA) and miRNA (microRNA,
hereinafter abbreviated as "miRNA"). Small RNA is known to function
as a guide molecule for finding target sequences in processes such
as translational suppression, mRNA degradation, or alteration of
chromatin structure. Small RNAs function via RNA interference
(RNAi) or miRNA molecular mechanisms. In addition, small RNA is
also known to play an important role in the regulation of
developmental processes (for example, as general remarks, refer to
Jikken Igaku (Experimental Medicine), 24, pp. 814-819, 2006; and
microRNA Jikken Purotokoru (microRNA Experimental Protocol), pp.
20-35, 2008, YODOSHA CO., LTD., herein incorporated by reference in
their entireties).
[0007] ES cell-specific microRNAs have been identified (Houbaviy et
al., Developmental Cell 5:351-58, 2003). In particular, ES
cell-specific expression of a microRNA cluster, which includes
several types of miRNAs in mouse ES cells, has been reported
(Houbaviy et al., Developmental Cell 5:351-58, 2003, herein
incorporated by reference in its entirety). It has also been
reported that miRNA-295 suppressed the expression of Rb12, a member
of the Rb tumor suppressor gene family, and increased the
expression of methylase to be thereby associated with DNA
methylation (Sinkkonen et al., Nature Structural & Molecular
Biology 15:259-267, 2008; Benetti et al., Nature Structural &
Molecular Biology 15:268-279, 2008, herein incorporated by
reference in their entireties). However, these documents do not
disclose any role of small RNA in the nuclear reprogramming of
somatic cells.
SUMMARY OF THE INVENTION
[0008] The present invention relates to methods for efficiently
preparing induced pluripotent stem cells. The present invention
provides methods for achieving efficient preparation of induced
pluripotent stem cells in the presence of miRNA. The present
invention also provides methods for efficient preparation of
induced pluripotent stem cells with a nuclear reprogramming factor.
The present invention also provides methods for efficient
preparation of induced pluripotent stem cells with a nuclear
reprogramming factor in the presence of increased miRNA as compared
to the level present in the somatic cell prior to nuclear
reprogramming. The present invention also provides such methods
wherein the nuclear reprogramming factor does not include c-Myc
and/or Sox2.
[0009] The invention provides a method of preparing induced
pluripotent stem cells, comprising nuclear reprogramming at least
one somatic cell with nuclear reprogramming factor and at least one
miRNA, wherein the at least one miRNA increases efficiency of the
nuclear reprogramming of the at least one somatic cell compared to
nuclear reprogramming of the at least one somatic cell with the
nuclear reprogramming factor in the absence of the at least one
miRNA.
[0010] The invention also provides such a method, wherein the at
least one miRNA is expressed in embryonic stem cells at a higher
level than in somatic cells.
[0011] The invention also provides such a method, wherein a gene
encoding the nuclear reprogramming factor and/or the at least one
miRNA is introduced into the at least one somatic cell.
[0012] The invention also provides such a method, wherein a vector
comprising the gene and/or a vector encoding the at least one miRNA
is introduced into the at least one somatic cell.
[0013] The invention also provides such a method, wherein the
vector comprising the gene or encoding the at least one miRNA is a
retroviral vector.
[0014] The invention also provides such a method, wherein the gene
is selected from an Oct family gene, a Klf family gene, and a Sox
family gene.
[0015] The invention also provides such a method, wherein the gene
is selected from Oct3/4, Klf4, and Sox2.
[0016] The invention also provides such a method, wherein the
nuclear reprogramming factor comprises Oct3/4, Klf4, and Sox2.
[0017] The invention also provides such a method, wherein the at
least one miRNA is introduced into the at least one somatic cell as
primary miRNA.
[0018] The invention also provides such a method, wherein the at
least one miRNA is introduced into the at least one somatic cell as
pre-miRNA.
[0019] The invention also provides such a method, wherein the at
least one miRNA comprises at least one miRNA represented by SEQ ID
NOs: 1 to 14.
[0020] The invention also provides such a method, wherein the at
least one miRNA comprises at least one miRNA contained in miRNA
cluster hsa-miR-302-367 cluster.
[0021] The invention also provides such a method, wherein the at
least one miRNA regulates DNA methylation.
[0022] The invention also provides such a method, wherein the at
least one miRNA regulates de novo DNA methylation.
[0023] The invention also provides such a method, wherein the at
least one miRNA down-regulates DNA methylation.
[0024] The invention also provides such a method, wherein the at
least one miRNA comprises at least 10 contiguous nucleotides of at
least one miRNA represented by SEQ ID NOs: 1 to 14.
[0025] The invention also provides such a method, wherein the at
least one miRNA comprises at least 30 contiguous nucleotides of at
least one miRNA represented by SEQ ID NOs: 1 to 14.
[0026] The invention also provides such a method, wherein the at
least one miRNA comprises at least 60 contiguous nucleotides of at
least one miRNA represented by SEQ ID NOs: 1 to 14.
[0027] The invention also provides such a method, wherein the
nuclear reprogramming factor does not include c-Myc and/or
Sox2.
[0028] The invention also provides such a method, wherein the at
least one miRNA comprises hsa-miR-302-367 cluster miRNA.
[0029] The invention also provides such a method, wherein the
nuclear reprogramming factor comprises an Oct family gene member, a
Sox family gene member, and a Klf family gene member.
[0030] The invention also provides such a method, wherein the at
least one miRNA comprises mmu-miR-295/295* and
mmu-miR-294/294*.
[0031] The invention also provides such a method, wherein the at
least one miRNA comprises hsa-miR-302-367 cluster, hsa-miR-371-373
cluster and hsa-miR-520c miRNA.
[0032] The invention also provides such a method, wherein the
nuclear reprogramming factor comprises a Klf family gene, and an
Oct family gene.
[0033] The invention also provides such a method, wherein the
nuclear reprogramming factor further comprises a Myc family
gene.
[0034] The invention also provides such a method, wherein the
nuclear reprogramming factor further comprises a Sox family
gene.
[0035] The invention also provides such a method, wherein the
nuclear reprogramming factor further comprises a Sox family
gene.
[0036] The invention also provides such a method, wherein the
nuclear reprogramming factor comprises KLF4 and OCT3/4.
[0037] The invention also provides such a method, wherein the
nuclear reprogramming factor excludes a Sox family gene.
[0038] The invention also provides such a method, wherein the
nuclear reprogramming factor excludes a Myc family gene.
[0039] The invention also provides such a method, where the at
least one somatic cell comprises a plurality of somatic cells.
[0040] The invention also provides a method of increasing the
efficiency of nuclear reprogramming comprising: adding a nuclear
reprogramming factor and at least one miRNA to at least one somatic
cell so that the number of induced pluripotent stem cells produced
is greater than in the absence of the added miRNA.
[0041] The invention also provides an induced pluripotent stem cell
induced by reprogramming a somatic cell, wherein the reprogramming
is performed by adding at least one miRNA and in the absence of
eggs, embryos, or embryonic stem (ES) cells.
[0042] The invention also provides such an induced pluripotent stem
cell, wherein the induced pluripotent stem cell is a human
cell.
[0043] The invention also provides an induced pluripotent stem cell
obtained by a method of preparing induced pluripotent stem cells,
comprising nuclear reprogramming at least one somatic cell with
nuclear reprogramming factor and at least one miRNA, wherein the at
least one miRNA increases efficiency of the nuclear reprogramming
of the at least one somatic cell compared to nuclear reprogramming
of the at least one somatic cell with the nuclear reprogramming
factor in the absence of the at least one miRNA.
[0044] The invention also provides an pluripotent stem cell
obtained by a method of increasing the efficiency of nuclear
reprogramming comprising: adding a nuclear reprogramming factor and
at least one miRNA to at least one somatic cell so that the number
of induced pluripotent stem cells produced is greater than in the
absence of the added miRNA.
[0045] The invention also provides somatic cell derived by inducing
differentiation of any of the above pluripotent stem cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] FIG. 1 shows the results of confirmation on the production
efficiency of induced pluripotent stem cells through induction of
nuclear reprogramming in mouse embryonic fibroblasts with a
combination of three genes comprising Oct3/4, Klf4, and Sox2 (this
combination--represented as "3f", "c-Myc(-)", or OSK"--means that
c-Myc was omitted from a combination of four genes comprising
Oct3/4, Klf4, Sox2 and c-Myc, which is highly efficient for nuclear
reprogramming), in the presence of various miRNAs. 3f+DsRed
represents a combination where DsRed (Discosoma sp. red fluorescent
protein) as a control was added to the combination of the
aforementioned three genes. The results of three independent
experiments are shown. The graph shows the number of ES-like
colonies in the cells transduced with OSK with or without DsRed, or
with various miRNAs.
[0047] FIG. 2 shows the production efficiency of induced
pluripotent stem cells. The top row of images shows the results of
nuclear reprogramming in mouse tail tip fibroblasts (TTFs) when
DsRed was added, as a control, to the combination of three genes
comprising Oct3/4, Klf4, and Sox2 (a combination of three genes in
which c-Myc was omitted from the combination of four genes). The
bottom row of images shows the results of induction of nuclear
reprogramming in mouse TTFs with the combination of three genes
comprising Oct3/4, Klf4, and Sox2 in the presence of mmu-miR-295.
The number in the figure indicates the number of Nanog GFP positive
colonies/the number of total colonies on days 7, 21, and 28 after
drug selection was started.
[0048] FIG. 3 shows the results of confirmation on the production
efficiency of induced pluripotent stem cells through induction of
nuclear reprogramming in adult human dermal fibroblasts expressing
the mouse ecotropic virus receptor Slc7a1 gene using lentivirus
(aHDF-Slc-7a1) with the combination of three genes comprising
Oct3/4, Klf4, and Sox2 (Myc(-)3f: a combination of three genes in
which c-Myc was omitted from the combination of four genes
comprising Oct3/4, Klf4, Sox2, and c-Myc), or the combination of
four genes comprising Oct3/4, Klf4, Sox2, and c-Myc (Y4f), in the
presence of various miRNAs.
[0049] FIGS. 4A-B shows the results of ES-like colonies produced
after transduction with 4 factors, i.e., OCT3/4, SOX2, KLF4, and
c-MYC (OSMK), as well as with 3 factors, i.e., OCT3/4, SOX2, KLF4
in the presence of various miRNAs (OSK+). FIG. 4A shows the number
of human ES-like colonies obtained by transduction with 4 factors
(OSMK), and with 3 factors without c-MYC plus miRNAs (OSK+). FIG.
4B shows the morphology of ES-like colonies from a subset of the
samples counted in FIG. 4A.
[0050] FIG. 5 shows expression of ES cell markers in iPS cells
produced by nuclear reprogramming of mouse Tail Tip Fibroblasts
(TTFs) with 4 factors (OCT3/4, SOX2, c-MYC, and KLF4) and with 3
factors (OCT3/4, SOX2, and KLF4, i.e. "Myc(-)3f")+mmu-miR-295/295*
or DsRed.
[0051] FIGS. 6A-B show the results of MEFs infected with 3 factors
(Oct3/4, c-MycWT, and Klf4, i.e., "Sox(-)") with mmu-miR-290-295
cluster, 290-5p/290-3p(mmu-miR-290), 291a-5p/291a-3p(mmu-miR-291a),
292-5p/292-3p(mmu-miR-292), 293/293*(mmu-miR-293),
294/294*(mmu-miR-294) or 295/295*(mmu-miR-295). FIG. 6A shows the
number of Nanog GFP positive colonies. FIG. 6B shows expression of
ES marker genes in iPS cells checked with RT-PCR.
[0052] FIGS. 7A-C show the results of iPS induction with Fb-Ng MEFs
(MEFs derived from Fbx 15-.beta. geo/Nanog-IRES-Puro.sup.r reporter
mouse) over-expressing Oct3/4, c-Myc, and Klf4
("Sox(-)")+mmu-miR-295/295* or hsa-miR-302-367 cluster miRNAs. FIG.
7A shows cell morphology of MEFs transduced with Oct3/4, c-Myc, and
Klf4 ("Sox(-)")+mmu-miR-295/295*. FIG. 7B shows chimeras derived
from iPS cells induced with Sox(-)3f+mmu-miR-295/295*. FIG. 7C
shows embryoid body (EB)-mediated in vitro differentiation by human
iPS cells. Human iPS cells (61B1, 61N2) were established by
transduction of 4 genes (OCT3/4, KLF4, SOX2, and c-MYC, i.e.,
"OSMK") or 3 genes (OCT3/4, KLF4, and c-MYC, i.e., "OMK(SOX(-)") in
the presence of hsa-miR-302-367 cluster miRNA. After culturing for
16 days, immunohistochemistry analysis was performed in the cells
by using an antibody against each of a-fetoprotein (AFP) which is a
differentiation marker for endodermal cells, .alpha.-smooth muscle
actin (a-SMA) which is a differentiation marker for mesodermal
cells, and GFAP (DAKO) which is a differentiation marker for
ectodermal cells. Nuclei were stained with Hoechst 33342
(Invitrogen).
[0053] FIG. 8 shows cell morphology of iPS cells induced with OSMK;
SOX(-) (OKM)+hsa-miR-302-367 cluster miRNA; and
OCT3/4+KLF4+hsa-miR-302-367 cluster miRNA.
[0054] TABLE 3 shows iPS induction by transduction with 4 factors
(OCT3/4, SOX2, MYC, KLF4, i.e., "OSMK"), with 3 factors
(SOX(-)3factors, i.e., "OMK") plus various miRNAs (OMK:mock or
miRNAs=2.5:1.5), and with 2 factors (OCT3/4+KLF4, i.e., "OK") plus
various miRNAs. Transduction with DsRed was performed as a as
control. On Day 40 after infection, the number of ES-like colonies
was counted. TABLE 3 shows the results of six independent
experiments (Exp. 54, 61, 63, 114, 130 and 133).
DETAILED DESCRIPTION OF THE INVENTION
[0055] The present invention provides methods for efficiently
preparing induced pluripotent stem cells through reprogramming of
one or more somatic cells. In particular, the present invention
provides efficient preparation of induced pluripotent stem cells in
the presence of miRNA. The present invention also provides
efficient preparation with or without using a suspected tumorigenic
factor: c-Myc. The present invention also provides efficient
preparation with or without using Sox2. The nuclear reprogramming
is preferably performed without c-Myc and/or Sox2.
[0056] The inventors of the present invention have conducted
intensive studies, and as a result, they have found that induced
pluripotent stem cells can be efficiently prepared by introduction
of nuclear reprogramming-inducing gene(s) into somatic cells in the
presence of specific miRNA. The present invention was achieved on
the basis of the above findings.
[0057] The present invention thus provides a method of preparing
induced pluripotent stem cells, comprising nuclear reprogramming
with a nuclear reprogramming factor in the presence of miRNA,
wherein said miRNA has a property of providing a higher nuclear
reprogramming efficiency in the presence of said miRNA than in the
absence thereof.
[0058] A preferred embodiment of the present invention provides the
aforementioned method wherein: (a) said miRNA is expressed in
embryonic stem cells at a higher level than in somatic cells;
and/or (b) said miRNA has a property of providing a higher nuclear
reprogramming efficiency in the presence of said miRNA than in the
absence thereof and/or (c) said nuclear reprogramming is performed
in the presence of increased levels of one or more miRNAs as
compared to the level(s) present in the somatic cell prior to
nuclear reprogramming.
[0059] Another preferred embodiment of the present invention
provides: the aforementioned method wherein the nuclear
reprogramming factor is either a single substance, or a combination
of a plurality of substances, which is/are positive in the
screening method of nuclear reprogramming factor described in
International Publication No. WO2005/80598 A1, incorporated by
reference herein in its entirety; the aforementioned method wherein
the nuclear reprogramming factor is either a gene product of a
single gene, or a combination of gene products of a plurality of
genes, which is/are positive in the screening method of nuclear
reprogramming factor described in International Publication No.
WO2005/80598 A1, incorporated by reference herein in its entirety;
the aforementioned method wherein the nuclear reprogramming with
the nuclear reprogramming factor is carried out by introduction of
the aforementioned gene(s) and/or substance(s) into somatic cells;
the aforementioned method wherein introduction of the
aforementioned gene(s) into somatic cells is carried out with a
recombinant vector; and the aforementioned method wherein the
nuclear reprogramming with the nuclear reprogramming factor is
carried out by introduction of gene product(s) of the
aforementioned gene(s) into somatic cells.
[0060] Yet another preferred embodiment of the present invention
provides the aforementioned method wherein: the gene encoding the
reprogramming factor comprises one or more gene(s) selected from an
Oct family gene, a Klf family gene, a Sox family gene, a Myc family
gene, a Lin family gene, and a Nanog gene, preferably a combination
of two genes selected from the aforementioned genes except for the
Myc family genes of Sox family genes, more preferably a combination
of three genes, and particularly preferably a combination four or
more genes. In a preferred embodiment, the combination can be any
combination of factors which does not comprise c-Myc or Sox2.
[0061] More preferable combinations are: (a) a combination of two
genes comprising of an Oct family gene and a Sox family gene; (b) a
combination of three genes comprising an Oct family gene, a Klf
family gene, and a Sox family gene; and (c) a combination of four
genes comprising an Oct family gene, a Sox family gene, a Lin
family gene, and a Nanog gene. Further, it is also preferable to
combine any of the above genes with a TERT gene and/or a SV40 Large
T antigen gene. It may be preferable to omit Klf family genes
depending on the situation. The Myc family genes may or may not be
included in these combinations. Combinations without the Myc family
gene can be suitably used according to the present invention.
[0062] Among these embodiments, particularly preferable
combinations are: a combination of two genes comprising Oct3/4 and
Sox2; a combination of three genes comprising Oct3/4, Klf4, and
Sox2; and a combination of four genes comprising Oct3/4, Sox2,
Lin28, and Nanog. It is also preferable to combine any of the above
genes with a TERT gene and/or a SV40 Large T antigen gene. It may
be preferable to omit Klf4 depending on the situation. c-Myc may be
included in these combinations. However, combinations without c-Myc
can be suitably used in the present invention.
[0063] Other preferable combinations are: (a) a combination of two
genes comprising of an Oct family gene and a Klf family gene; (b) a
combination of three genes comprising an Oct family gene, a Klf
family gene, and a Myc family gene. Yet another preferred
embodiment of the present invention provides: the aforementioned
method wherein the somatic cells are those derived from mammals
including human, mouse, rat, cattle, sheep, horse, monkey, and
hamster, preferably somatic cells from human or mouse, and most
preferably somatic cells from human; the aforementioned method
wherein the somatic cells are human embryonic cells, or adult
human-derived somatic cells; and the aforementioned method wherein
the somatic cells are somatic cells collected from a patient.
[0064] Yet another preferred embodiment of the present invention
provides the aforementioned method wherein the miRNA comprises one
or more miRNA(s) included in the RNA sequences specified by the
registration names of the miRBase database or the accession numbers
shown in Table 1 or Table 2; the aforementioned method wherein the
RNA sequences specified by the registration names of the miRBase
database (and the accession numbers) shown in Table 1 or Table 2
comprise one or more RNA(s) selected from hsa-miR-372 (MI0000780),
hsa-miR-373 (MI0000781), hsa-miR-302b (MI0000772), hsa-miR-302c
(MI0000773), hsa-miR-302a (MI0000738), hsa-miR-302d (MI0000774),
hsa-miR-367 (MI0000775), hsa-miR-520c (MI0003158), mmu-miR-290
(MI0000388), mmu-miR-291a (MI0000389), mmu-miR-294 (MI0000392), and
mmu-miR-295 (MI0000393); the aforementioned method wherein the
miRNA comprises miRNA included in RNA specified by hsa-miR-302-367;
and the aforementioned method wherein the miRNA comprises one or
more miRNA(s) included in one or more RNA sequence(s) selected from
the RNA sequences represented by SEQ IDS: 1 to 14 in the Sequence
Listing.
[0065] The present invention provides an oligonucleotide comprising
at least 10 contiguous nucleotides in the nucleotide sequence of
the miRNA of the present invention, and an antisense
oligonucleotide having a sequence that is complementary to that of
the above oligonucleotide. The present invention also provides an
oligonucleotide comprising at least 15, at least 20, at least 30,
at least 50, or at least 60 contiguous nucleotides in the
nucleotide sequence of the miRNA of the present invention. The
present invention also provides an oligonucleotide comprising at
least 70, at least 80, at least 100, at least 150, at least 200, at
least 300, at least 400, at least 600, or at least 800 contiguous
nucleotides in the nucleotide sequence of the miRNA of the present
invention.
[0066] The present invention also provides induced pluripotent stem
cells that can be obtained by the aforementioned method. In
addition, the present invention also provides somatic cells
obtained by inducing differentiation from the abovementioned
induced pluripotent stem cells.
[0067] Further, the present invention provides a stem cell therapy
comprising transplanting somatic cells into a patient, wherein the
somatic cells are obtained by inducing differentiation from induced
pluripotent stem cells that are obtained according to the
aforementioned method by using somatic cells isolated and collected
from a patient.
[0068] In addition, the present invention provides a method for
evaluation of physiological effect or toxicity of a compound, a
drug, or a toxic agent, with use of various cells obtained by
inducing differentiation from induced pluripotent stem cells that
are obtained by the aforementioned method.
[0069] Further, the present invention provides: a method for
preparing induced pluripotent stem cells which uses miRNA expressed
in embryonic stem cells at a higher level than in somatic cells,
and having a property of providing a higher nuclear reprogramming
efficiency in the presence of said miRNA than in the absence
thereof; and a nuclear reprogramming method of somatic cells which
uses miRNA expressed in embryonic stem cells at a higher level than
in somatic cells, and having a property of providing a higher
nuclear reprogramming efficiency in the presence of said miRNA than
in the absence thereof.
[0070] In addition, the present invention provides methods
comprising the use of miRNA expressed in embryonic stem cells at a
higher level than in somatic cells (e.g., the miRNA may be
expressed at levels which are higher in the ES cell as compared to
the ES cell which has differentiated or which has begun
differentiating such as determined by RT-PCR or Northern blot
analysis), and having a property of providing a higher nuclear
reprogramming efficiency in the presence of said miRNA than in the
absence thereof, for preparation of induced pluripotent stem cells;
and methods relating to the use of miRNA expressed in embryonic
stem cells at a higher level than in somatic cells, and having a
property of providing a higher nuclear reprogramming efficiency in
the presence of the miRNA than in the absence thereof, for nuclear
reprogramming of somatic cells. In other words, nuclear
reprogramming, and thus, induced pluripotent stem cell production,
can be performed in the presence of miRNA and in the absence of
miRNA. The nuclear reprogramming may also be performed in the
presence of various amounts and/or kinds of miRNA, such that, for
example, the efficiency of the nuclear reprogramming is increased
when the level of the miRNA is increased in the somatic cell prior
to nuclear reprogramming.
[0071] In addition, the present invention provides methods
comprising the use of miRNA having a property of providing a higher
nuclear reprogramming efficiency in the presence of said miRNA than
in the absence thereof, for preparation of induced pluripotent stem
cells. For example, the presence of added miRNA can provide the
formation of an induced pluripotent stem cell as compared to the
lack of formation in the absence of the miRNA. Also, for example
when nuclear reprogramming is performed on the same number of
somatic cells in the presence of a nuclear reprogramming factor
containing the same components in the same concentrations with and
without addition of miRNA, increased efficiency can be observed
when a greater number of induced pluripotent stem cells are
generated in the sample which comprises the addition of miRNA than
in the sample without the addition of miRNA. In another embodiment,
increased efficiency of induced pluripotent stems cell production
can also be achieved with increased amounts of miRNA as compared to
miRNA amounts present in the somatic cell prior to nuclear
reprogramming.
BEST MODE FOR CARRYING OUT THE INVENTION
[0072] The methods of the present invention relate to, e.g., a
method for preparing induced pluripotent stem cells, comprising
nuclear reprogramming with a nuclear reprogramming factor in the
presence of miRNA, wherein said miRNA has a property of providing a
higher nuclear reprogramming efficiency in the presence of said
miRNA than in the absence thereof. In a preferred embodiment of the
present invention, (a) said miRNA is expressed in embryonic stem
cells at a higher level than in somatic cells; and (b) said RNA has
a property of providing a higher nuclear reprogramming efficiency
in the presence of said miRNA than in the absence thereof.
[0073] As for the miRNA, for example, its classification and in
vivo functions are described in Jikken Igaku (Experimental
Medicine), 24, pp. 814-819, 2006; microRNA Jikken Purotokoru
(microRNA Experimental Protocol), pp. 20-35, 2008, YODOSHA CO.,
LTD. The number of nucleotides of miRNA is for example 18 to 25,
and preferably about 19 to 23. At present, a database storing data
relating to about 1,000 miRNA sequences is available (for example,
miRBase, Griffiths-Jones et al. Nucleic Acids Research
36:D154-D158, 2008 (published online Nov. 8, 2007), see also
http://microrna.sanger.ac.uk/sequences/index.shtml [online]), and
it is possible for those skilled in the art to obtain any miRNA
data therefrom, and to readily extract miRNA expressed in embryonic
stem cells at a higher level than in somatic cells. In addition, it
is also possible to readily specify miRNA expressed in embryonic
stem cells at a higher level than in somatic cells by confirming
the difference in miRNA expression between embryonic stem cells and
somatic cells with use of available techniques for those skilled in
the art such as miRNA microarray and real-time PCR analyses.
[0074] The difference in the nuclear reprogramming efficiency with
and without miRNA can be understood by the following manner, as
specifically described in Examples of this application: transgenic
mice are generated by insertion of sequences encoding Enhanced
Green Fluorescent Protein (EGFP) and a puromycin resistance gene
downstream of a Nanog gene promoter region, the expression of which
is specific to ES cells; then, three genes, for example, Oct3/4,
Sox2, and Klf4, and various miRNAs are introduced into embryonic
fibroblasts derived from these transgenic mice to induce nuclear
reprogramming, followed by confirmation of the production
efficiency of induced pluripotent stem cells. The production
efficiency can be determined, for example, by counting the number
of colonies. More specifically, the number of colonies can be
compared by the following manner: drug selection is started from
the 21st day after introduction of the above genes and miRNA; and
the number of total colonies and the number of Nanog GFP positive
colonies (GFP, the expression of which is induced by the Nanog gene
promoter region, is observable under fluorescent microscopy) are
counted on the 28th day. It should be understood, however, that:
the confirmation of the nuclear reprogramming efficiency is not
limited to the above method; appropriate modification and
alteration can be made in the above method; and any appropriate
method can be employed by those skilled in the art.
[0075] As for the miRNA, it is preferable to use miRNA derived from
the same animal species as the target animal whose somatic cells
are to be reprogrammed. Usable miRNA includes wild type miRNA as
well as miRNAs in which one to several nucleotides (for example 1
to 6 nucleotides, preferably 1 to 4 nucleotides, more preferably 1
to 3 nucleotides, yet more preferably 1 or 2 nucleotides, and most
preferably 1 nucleotide) are substituted, inserted, and/or deleted,
and which are capable of exerting equivalent functions to those of
the wild type miRNA in vivo. For example, the miRNA of the present
invention includes miRNAs in which one to several nucleotides are
substituted, inserted, and/or deleted, and which increase the
efficiency of iPS cell production. The miRNA of the present
invention also includes miRNAs in which one to several nucleotides
are substituted, inserted, and/or deleted, and which improve the
efficiency of nuclear reprogramming. The miRNA of the present
invention also includes miRNAs in which one to several nucleotides
are substituted, inserted, and/or deleted, and which regulate DNA
methylation. The present invention also includes such miRNAs
wherein the DNA methylation is down-regulated. The present
invention also includes such miRNAs wherein the DNA methylation is
de novo DNA methylation.
[0076] Examples of the miRNA preferably used in the methods of the
present invention can include, but are not limited to, one or more
miRNA(s) included in the following RNA sequences registered in the
miRBase: hsa-miR-372 (MI0000780), hsa-miR-373 (MI0000781),
hsa-miR-302b (MI0000772), hsa-miR-302c (MI0000773), hsa-miR-302a
(MI0000738), hsa-miR-302d (MI0000774), hsa-miR-367 (MI0000775),
hsa-miR-520c (MI0003158), mmu-miR-290 (MI0000388), mmu-miR-291a
(MI0000389), mmu-miR-294 (MI0000392), and mmu-miR-295 (MI0000393)
(Numbers in the brackets respectively indicate miRBase accession
numbers. The symbol "hsa-miR-" represents human miRNA, and the
symbol "mmu-miR-" represents mouse miRNA.).
[0077] In the method of the present invention, miRNAs that have
been confirmed to improve the nuclear reprogramming efficiency in
the above manner can be used either alone or in combinations of two
or more types. In addition, a plurality of miRNAs forming a cluster
may also be used. For example, hsa-miR-302-367 which is available
as a miRNA cluster, or individual miRNAs from the hsa-miR-302-367
cluster, and the like may be used. Examples of RNA sequences for
use in the present invention are shown in SEQ IDS: 1 to 14 in the
Sequence Listing. SEQ ID: 1: mmu-miR-294 (MI0000392); SEQ ID: 2:
mmu-miR-295 (MI0000393); SEQ ID: 3: hsa-miR-372 (MI0000780); SEQ
ID: 4: hsa-miR-373 (MI0000781); SEQ ID: 5: hsa-miR-302b
(MI0000772); SEQ ID: 6: hsa-miR-302c (MI0000773); SEQ ID: 7:
hsa-miR-302a (MI0000738); SEQ ID: 8: hsa-miR-302d (MI0000774); SEQ
ID: 9: hsa-miR-367 (MI0000775); SEQ ID: 10: hsa-miR-520c
(MI0003158); SEQ ID: 11: mmu-miR-291a (MI0000389); SEQ ID:13:
mmu-miR-290 (MI0000388), and SEQ ID:14: hsa-miR-371-373 cluster. In
addition, RNA represented by SEQ ID: 12: hsa-miR-302-367 cluster
can also be preferably used. Among these RNA sequences, some RNA
sequences may include a plurality of miRNAs within one sequence.
Use of such an RNA sequence may achieve efficient production of iPS
cells. Further, an RNA sequence including a plurality of miRNAs
within one sequence and one or more other RNA sequence(s) including
one or more miRNA(s) can also be used in combination.
[0078] miRNA is non-coding RNA which is not translated into a
protein. miRNA is first transcribed as pri-miRNA from a
corresponding gene, then this pri-miRNA generates pre-miRNA having
a characteristic hairpin structure of about 70 nucleotides, and
this pre-miRNA is further processed into mature miRNA, which is
mediated by Dicer. In the present invention, not only mature miRNA
but also pri-miRNA or pre-miRNA can be used as long as the effect
of the present invention is not impaired. In addition, miRNA for
use in the present invention may be either natural type or
non-natural type. Thus, any small RNA or RNA precursor may be used
as long as the effect of the pre/sent invention is not
impaired.
[0079] The production method of miRNA for use in the present
invention is not specifically limited, although the production can
be achieved, for example, by a chemical synthetic method or a
method using genetic recombination technique. When the production
is carried out by a method using genetic recombination technique,
miRNA for use in the present invention can, for example, be
produced through transcription reaction with use of a DNA template
and a RNA polymerase obtained by means of gene recombination.
Examples of usable RNA polymerase include a T7 RNA polymerase, a T3
RNA polymerase, and a SP6 RNA polymerase.
[0080] Alternatively, a recombinant vector capable of expressing
miRNA can be produced by insertion of miRNA-encoding DNA into an
appropriate vector under the regulation of expression control
sequences (promoter and enhancer sequences and the like). The type
of vector used herein is not specifically limited, although DNA
vectors are preferred. Examples thereof can include viral vectors
and plasmid vectors. The viral vector is not specifically limited,
although retroviral vectors, adenoviral vectors, adeno-associated
viralvectors, and the like can be employed. In addition, as to the
above plasmids, mammalian expression plasmids well known to those
skilled in the art can be employed.
[0081] Methods for using a retrovirus as a vector are disclosed in
WO 2007/69666 A1; Takahashi et al., Cell 126:663-676, 2006; and
Takahashi et al., Cell 131:861-872, 2007, which are herein
incorportated by reference in their entireties. Methods for using a
lentivirus as a vector are disclosed in Yu et al., Science
318:1917-1920, 2007, which is herein incorporated by reference in
its entirety. Methods for using adenovirus as a vector are
disclosed in Stadtfeld et al., Science 322:945-949, 2008, which is
herein incorporated by reference in its entirety. Methods for using
a plasmid as a non-viral vector are disclosed in U.S. Provisional
Application No. 61/071,508; U.S. Provisional Application No.
61/136,246; U.S. Provisional Application No. 61/136,615; and U.S.
Provisional application Ser. No. ______ (Attorney Docket No.
V35667) entitled "Method for Nuclear Reprogramming" filed Nov. 21,
2008; and Okita et al., Science 322:949-953, 2008, which are herein
incorporated by reference in their entireties. One of ordinary
skill in the art could choose and use an appropriate method from
among the above known methods, or from any of the other known
methods or vectors available in the prior art.
[0082] Nuclear reprogramming can be performed in the presence of
miRNA in any number of ways. The manner of providing the miRNA is
not specifically limited, although examples thereof can include a
method for directly injecting miRNA into nuclei of somatic cells,
and a method for introducing an appropriate recombinant vector
capable of expressing miRNA into somatic cells. However, these
methods are not to be considered as limiting.
[0083] The method for introducing a recombinant vector into somatic
cells is not specifically limited, and can be carried out by any
method well known to those skilled in the art. Examples of the
employable methods can include transient transfection,
microinjection, a calcium phosphate precipitation method,
liposome-mediated transfection, DEAE dextran-mediated transfection,
electroporation, and methods comprising the use of a gene gun.
[0084] As to confirming a nuclear reprogramming factor, for
example, the screening method of nuclear reprogramming factor
described in International Publication No. WO2005/80598 A1,
incorporated by reference herein in its entirety, can be used.
Those skilled in the art are able to screen a nuclear reprogramming
factor for use in the method of the present invention by referring
to the above publication. In addition, the nuclear reprogramming
factor can also be confirmed by using a method in which appropriate
modification or alteration has been made in the above screening
method.
[0085] Examples of the combination of genes encoding reprogramming
factors are disclosed in International Publication No.
WO2007/069666 A1 and its family member U.S. patent application Ser.
No. 12/213,035 and U.S. patent application Ser. No. 12/289,873,
filed Nov. 6, 2008, entitled "Nuclear Reprogramming Factor and
Induced Pluripotent Stem Cells" which are incorporated by reference
herein in their entireties. Those skilled in the art are able to
appropriately select a gene that can be preferably used for the
method of the present invention by referring to the above
publication. In addition, other examples of the combinations of
genes encoding reprogramming factors are disclosed, for example, in
Yu et al., Science 318:1917-20, 2007, incorporated by reference
herein in its entirety. Accordingly, those skilled in the art are
able to understand the variety of the combination of genes encoding
reprogramming factors, and are able to employ an appropriate
combination of genes in the method of the present invention, which
combination is not disclosed in International Publication No.
WO2007/069666 A1 or Yu et al., Science 318:1917-20, 2007, by using
the screening method of nuclear reprogramming factor described in
International Publication No. WO2005/80598 A1.
[0086] Examples of the gene encoding a reprogramming factor that
can be used for the method of the present invention can include:
one or more gene(s) selected from an Oct family gene, a Klf family
gene, a Sox family gene, a Myc family gene, a Lin family gene, and
a Nanog gene; preferably one or more gene(s) selected from an Oct
family gene, a Klf family gene, a Sox family gene, a Lin family
gene, and a Nanog gene, and excluding a Myc family gene; one or
more gene(s) selected from an Oct family gene, a Klf family gene, a
Myc family gene, a Lin family gene, and a Nanog gene, and excluding
a Sox family gene; more preferably a combination of two genes; yet
more preferably a combination of three genes; and most preferably a
combination of four genes.
[0087] Regarding the Oct family gene, Klf family gene, Sox family
gene, and Myc family gene, specific examples of these family genes
are described in International Publication No. WO2007/069666 A1.
Regarding the Lin family gene, those skilled in the art are able to
extract the family gene in a similar way. Examples of the Lin
family genes include, for example, Lin28 and Lin28b. The NCBI
accession numbers of Lin28 are NM 145833 (mouse) and
NM.sub.--024674 (human). The NCBI accession numbers of Lin28b are
NM.sub.--001031772 (mouse) and NM.sub.--001004317 (human).
[0088] In addition, reprogramming factor(s) encoded by one or more
gene(s) selected from an Oct family gene, a Klf family gene, a Sox
family gene, a Myc family gene, a Lin family gene, and a Nanog
gene, may be substituted by, for example a cytokine, or one or more
other low molecular weight compound(s) in some cases. Examples of
such low molecular weight compound(s) can include low molecular
weight compounds having an enhancing action on the expression of
one or more gene(s) selected from an Oct family gene, a Klf family
gene, a Sox family gene, a Myc family gene, a Lin family gene, and
a Nanog gene. Those skilled in the art are able to readily screen
such low molecular weight compound(s).
[0089] More preferable combinations of genes are as follows:
(a) a combination of two genes comprising an Oct family gene and a
Sox family gene; (b) a combination of three genes comprising an Oct
family gene, a Klf family gene, and a Sox family gene; (c) a
combination of four genes comprising an Oct family gene, a Sox
family gene, a Lin family gene, and a Nanog gene; (d) a combination
of two genes comprising an Oct family gene and a Klf family gene;
and (e) a combination of three genes comprising an Oct family gene,
a Klf family gene, and a Myc family gene. However, these
combinations are not to be considered as limiting.
[0090] All of these genes are commonly present in mammals,
including human. In order to use the above genes according to the
present invention, genes derived from any mammal (for example,
derived from a mammal such as human, mouse, rat, cattle, sheep,
horse, and monkey) can be employed. In addition, it is also
possible to use a wild type gene product, as well as mutant gene
products in which several amino acids (for example 1 to 10 amino
acids, preferably 1 to 6 amino acids, more preferably 1 to 4 amino
acids, yet more preferably 1 to 3 amino acids, and most preferably
1 or 2 amino acids) have been substituted, inserted, and/or
deleted, and which have comparable equivalent functions to those of
the wild type gene product. For example, as to the c-Myc gene
product, a stable type variant, e.g., (T58A) and the like may also
be used as well as the wild type. The same principle can be applied
to other gene products.
[0091] In addition to the above genes, a gene encoding a factor
which induces immortalization of cells may also be combined. As
disclosed in International Publication No. WO2007/069666 A1, for
example, one or more gene(s) selected from a TERT gene, and
following genes: SV40 Large T antigen, HPV16 E6, HPV16 E7, and
Bmil, can be either solely used or jointly used in an appropriate
combination.
[0092] Preferable combinations are as follows, for example:
(e) a combination of four genes comprising an Oct family gene, a
Klf family gene, a Sox family gene, and a TERT gene; (f) a
combination of four genes comprising an Oct family gene, a Klf
family gene, a Sox family gene, and a SV40 Large T antigen gene;
and (g) a combination of five genes comprising an Oct family gene,
a Klf family gene, a Sox family gene, a TERT gene, and a SV40 Large
T antigen gene. The Klf family gene may be omitted from the above
combinations.
[0093] Further, in addition to the above genes, one or more gene(s)
selected from Fbx15, ERas, ECAT15-2, Tell, and .beta.-catenin may
be combined, and/or one or more gene(s) selected from ECAT1, Esg1,
Dnmt3L, ECAT8, Gdf3, Sox15, ECAT15-1, Fthl17, Sal14, Rex1, UTF1,
Stella, Stat3, and Grb2 may also be combined. These combinations
are specifically described in International Publication No.
WO2007/069666 A1.
[0094] Particularly preferable combinations of genes are as
follows:
(1) a combination of two genes comprising Oct3/4 and Sox2; (2) a
combination of three genes comprising Oct3/4, Klf4, and Sox2; (3) a
combination of four genes comprising Oct3/4, Sox2, Lin28, and
Nanog; (4) a combination of four genes comprising Oct3/4, Sox2,
TERT, and SV40 Large T antigen gene; (5) a combination of five
genes comprising Oct3/4, Klf4, Sox2, TERT, and SV40 Large T antigen
gene; (6) a combination of two genes comprising Oct3/4 and Klf4;
(7) a combination of three genes comprising Oct3/4, Klf4, and
c-Myc; and (8) a combination of four genes comprising Oct3/4, Sox2,
Klf4, and c-Myc. However, these combinations are not to be
considered as limiting.
[0095] The factors including the gene products as mentioned above
may also be combined with one or more gene product(s) of gene(s)
selected from: Fbx15, Nanog, ERas, ECAT15-2, Tell, and
.beta.-catenin. Further, these factors may also be combined with
one or more gene product(s) of gene(s) selected from: ECAT1, Esg1,
Dnmt3L, ECAT8, Gdf3, Sox15, ECAT15-1, Fthl17, Sal14, Rex1, UTF1,
Stella, Stat3, and Grb2, for example. These gene products are
disclosed in International Publication No. WO2007/069666 A1.
However, gene products that can be included in the nuclear
reprogramming factors of the present invention are not limited to
the gene products of genes specifically described above. The
nuclear reprogramming factors of the present invention can include
other gene products which can function as a nuclear reprogramming
factor, as well as one or more factors involving differentiation,
development, or proliferation, and factors having other
physiological activities. It should be understood that the
aforementioned aspect may also be included within the scope of the
present invention.
[0096] Among these genes, if one or more gene product(s) is/are
already expressed in somatic cells to be reprogrammed, such gene
products can be excluded from the factors to be introduced. For
example, one or more gene(s) besides the already-expressed gene(s)
can be introduced into somatic cells by an appropriate gene
introduction method, for example, a method using a recombinant
vector. Alternatively, among these genes, if one or more gene
product(s) is/are introduced into nuclei by a technique such as
addition of an HIV virus-derived TAT peptide and/or nuclear
localization signal to form a fusion protein or by a technique such
as nuclear microinjection, or simply by addition of a small
molecule capable of diffusing across the plasma membrane, the other
one or more gene(s) can be introduced by an appropriate gene
introduction method, for example, a method using a recombinant
vector.
[0097] In addition, a gene product serving as a nuclear
reprogramming factor may be either a protein itself produced from
the abovementioned gene, or in the form of a fusion gene product
between such a protein and another protein, a peptide, or the like.
For example, a fusion protein having Green Fluorescent Protein
(GFP) and a fusion gene product having a peptide such as a
histidine tag may also be used. Further, use of a prepared fusion
protein having a HIV virus-derived TAT peptide enables the
promotion of endocytosis of a nuclear reprogramming factor through
cell membrane, and also enables the induction of reprogramming by
simply adding such a fusion protein into the medium while avoiding
complicated manipulations such as gene introduction. The
preparation method of the aforementioned fusion gene product is
well known to those skilled in the art, and therefore those skilled
in the art are able to readily design and prepare an appropriate
fusion gene product according to the purpose.
[0098] In this application, the term "induced pluripotent stem
cells (iPS cells)" refers to cells having similar properties to
those of ES cells, and more specifically the term includes
undifferentiated cells which are reprogrammed from somatic cells
and have pluripotency and proliferation potency. However, this term
is not to be construed as limiting in any sense, and should be
construed to have its broadest meaning. The preparation method of
induced pluripotent stem cells with the use of a nuclear
reprogramming factor is described in International Publication No.
WO2005/80598 A1 (the term "ES-like cell" is used in this
publication), and methods for isolating induced pluripotent stem
cells are also specifically described. In addition, specific
examples of the reprogramming factor and specific examples of the
reprogramming method of somatic cells with use of such a
reprogramming factor are disclosed in International Publication No.
WO2007/069666 .mu.l. Accordingly, it is desirable for those skilled
in the art to refer to these publications for carrying out the
present invention.
[0099] The preparation method of induced pluripotent stem cells
from somatic cells by the method of the present invention is not
specifically limited, and any method can be employed as long as the
method enables nuclear reprogramming of somatic cells with a
nuclear reprogramming factor in the presence of miRNA in an
environment where somatic cells and induced pluripotent stem cells
can grow. For example, a vector comprising a gene which can express
a nuclear reprogramming factor can be used to introduce such a gene
into somatic cells, and at either the same or different timing, a
recombinant vector which can express miRNA can be introduced into
the somatic cells. If such vectors are used, two or more genes may
be incorporated into a vector to effect simultaneous expression of
respective gene products in somatic cells.
[0100] When gene(s) and/or miRNA are introduced into somatic cells
with use of a vector which can express the above gene(s), the
expression vector may be introduced into somatic cells that have
been cultured on feeder cells, or the expression vector may also be
introduced into somatic cells alone. The latter method is sometimes
more suitable in order to improve the introduction efficiency of
the expression vector. As to the feeder cells, there may be
appropriately used feeder cells for use in culture of embryonic
stem cells. Examples thereof can include primary culture cells of
14 or 15 day-mouse embryonic fibroblasts and STO cells of
fibroblast cell line, which are treated with either radiation or a
drug such as mitomycin C.
[0101] The culture of somatic cells introduced with a nuclear
reprogramming factor under an appropriate condition leads to
autonomous nuclear reprogramming, as a result of which induced
pluripotent stem cells can be produced from somatic cells. The
process for introducing a gene encoding a nuclear reprogramming
factor and/or miRNA into somatic cells with use of an expression
vector to thereby obtain induced pluripotent stem cells can be
performed in accordance with, for example, a method using a
retrovirus. Examples of such method include methods described in
publications such as Takahashi et al., Cell 126:663-76, 2006;
Takahashi et al., Cell 131:861-72, 2007; Yu et al., Science
318:1917-20, 2007. When human induced pluripotent stem cells are to
be produced, it is desirable to set the cell culture density after
the introduction of an expression vector to be lower than normal
cases for culturing animal cells. For example, it is preferable to
keep culturing at a density of 1.times.10.sup.4 to 1.times.10.sup.5
cells/10 cm dish, and more preferably about 5.times.10.sup.4
cells/10 cm dish. The medium for use in culture is not specifically
limited, and can be appropriately selected by those skilled in the
art, although for example it is sometimes preferable to use a
medium suitable for human ES cell culture for the production of
human induced pluripotent stem cells. The medium selection and
culture condition can be referred to the above publications.
[0102] Thus produced induced pluripotent stem cells can be checked
with various markers specific to undifferentiated cells, and the
means therefor is described in the above publications specifically
in detail. For example, some pluripotent cell markers include:
alkaline phosphatase (AP); ABCG2; stage specific embryonic
antigen-1 (SSEA-1); SSEA-3; SSEA-4; TRA-1-60; TRA-1-81;
Tra-2-49/6E; ERas/ECAT5, E-cadherin; .beta.III-tubulin;
.alpha.-smooth muscle actin (.alpha.-SMA); fibroblast growth factor
4 (Fgf4), Cripto, Dax1; zinc finger protein 296 (Zfp296);
N-acetyltransferase-1 (Nat1); (ES cell associated transcript 1
(ECAT1); ESG1/DPPA5/ECAT2; ECAT3; ECAT6; ECAT7; ECAT8; ECAT9;
ECAT10; ECAT15-1; ECAT15-2; Fthl17; Sal14; undifferentiated
embryonic cell transcription factor (Utf1); Rex1; p53; G3PDH;
telomerase, including TERT; silent X chromosome genes; Dnmt3a;
Dnmt3b; TRIM28; F-box containing protein 15 (Fbx15); Nanog/ECAT4;
Oct3/4; Sox2; Klf4; c-Myc; Esrrb; TDGF1; GABRB3; Zfp42, FoxD3;
GDF3; CYP25A1; developmental pluripotency-associated 2 (DPPA2); and
T-cell lymphoma breakpoint 1 (Tell); DPPA3/Stella; DPPA4. Other
markers can include Dnmt3L; Sox15; Stat3; Grb2; SV40 Large T
Antigen; HPV16 E6; HPV16 E7, 13-catenin, and Bmi1. Such cells can
also be characterized by the down-regulation of markers
characteristic of the differentiated cell from which the iPS cell
is induced. For example, iPS cells derived from fibroblasts may be
characterized by down-regulation of the fibroblast cell marker Thy1
and/or up-regulation of SSEA-3 and 4. It is understood that the
present invention is not limited to those markers listed herein,
and encompasses markers such as cell surface markers, antigens, and
other gene products including ESTs, RNA (including microRNAs and
antisense RNA), DNA (including genes and cDNAs), and portions
thereof.
[0103] Various media capable of retaining undifferentiation
property and pluripotency of ES cells and various media incapable
of retaining these properties are known in the art, and appropriate
combination of these media enables efficient isolation of induced
pluripotent stem cells. The differentiation ability and
proliferation potency of thus isolated induced pluripotent stem
cells can be readily checked by those skilled in the art, with use
of general checking means for ES cells. In addition, colonies of
induced pluripotent stem cells can be obtained by growing thus
produced induced pluripotent stem cells under an appropriate
condition, and the presence of these induced pluripotent stem cells
can be identified with reference to the shape of their colonies.
For example, it is known that mouse induced pluripotent stem cells
form raised colonies, while human induced pluripotent stem cells
form flat colonies. These colony shapes are respectively very
similar to those of mouse ES cells and human ES cells, and those
skilled in the art are thus able to identify these produced induced
pluripotent stem cells with reference to the shape of their
colonies.
[0104] The type of somatic cell to be reprogrammed by the method of
the present invention is not specifically limited, and any somatic
cell can be used. For example, somatic cells derived from any
mammal (for example, derived from a mammal such as human, mouse,
rat, cattle, sheep, horse, and monkey) can be employed. Not only
embryonic somatic cells but also neonatal somatic cells, matured
somatic cells, and tissue stem cells may also be used. In addition,
various somatic cells such as skin cells, liver cells, and gastric
mucosa cells can be reprogrammed. For use of induced pluripotent
stem cells in therapies against diseases, it is desirable to use
somatic cells isolated from the patient. For example, somatic cells
involved in a disease and somatic cells associated with a therapy
for a disease can be used.
[0105] The application of induced pluripotent stem cells produced
by the method of the present invention is not specifically limited,
and these cells can be used for every examination/study to be
performed with use of ES cells, and for any disease therapy which
utilizes ES cells. For example, induced pluripotent stem cells
obtained by the method of the present invention can be induced into
desired differentiated cells (such as nerve cells, myocardial
cells, blood cells and insulin-producing cells) by treatment with
retinoic acid, a growth factor such as EGF, or glucocorticoid, so
that appropriate tissue can be formed. Stem cell therapies through
autologous cell transplantation can be achieved by returning these
differentiated cells or tissue obtained in the above manner, into
the patient. However, the application of the induced pluripotent
stem cells of the present invention is not to be limited to the
abovementioned specific aspects.
EXAMPLES
[0106] The present invention will be explained more specifically
with reference to the following examples. However, the scope of the
present invention is not limited to these examples.
Example 1
Preparation of Induced Pluripotent Stem Cells Through Nuclear
Reprogramming of Mouse Embryonic Fibroblasts
[0107] pMXs-based retroviral vectors, which respectively encode
each of three genes of mouse-derived Oct3/4, Sox2, and Klf4,
control DsRed or each miRNA of 18 types of miRNAs, were transfected
into PLAT-E cells using FuGENE 6 reagent (Roche) to get
retroviruses. On the next day, embryonic fibroblasts (Nanog GFP
MEF, WO2007/069666 A1) derived from transgenic mice generated by
insertion of sequences encoding EGFP gene and puromycin resistance
gene downstream of a Nanog gene promoter region, were seeded at
1.times.10.sup.5 cells/well in 6-well plates. On the next day,
these cells were infected with retroviruses expressing Oct3/4,
Sox2, Klf4, and each type of miRNA selected from 18 types of
miRNAs, at a ratio of 1 ml of virus mixture expressing these three
factors to 1 ml of virus solution expressing miRNA or DsRed, so as
to prepare induced pluripotent stem cells through nuclear
reprogramming.
TABLE-US-00001 TABLE 1 miRNA sequence (other name(s) miRBase miRNA
number indicated in parentheses) accession number 1 mmu-miR-150
MI0000172 2 mmu-miR-182 MI0000224 3 mmu-miR-126 MI0000153 4
mmu-miR-290-295 cluster 5 mmu-miR-290 MI0000388
(mmu-miR-290-5p/290-3p) 6 mmu-miR-291a MI0000389
(mmu-miR-291a-5p/291a-3p) 7 mmu-miR-292 MI0000390
(mmu-miR-292-5p/292-3p) 8 mmu-miR-294 MI0000392 (mmu-miR-294/294*)
X(9) mmu-miR-295 MI0000393 (mmu-miR-295/295*) 10 mmu-miR-17-92
cluster 11 mmu-miR-323 MI0000592 12 mmu-miR-130b MI0000408 13
mmu-miR-7a-1 MI0000728 14 mmu-miR-7a-2 MI0000729 15 mmu-miR-205
MI0000248 16 mmu-miR-200a MI0000554 17 mmu-miR-200c MI0000694 18
mmu-miR-mix *indicates star form of miRNA.
[0108] From the third day after infection, the cells were cultured
in an ES cell medium containing LIF. On the fourth day after
infection, the cells were harvested by trypsinization, and the
whole amount thereof was spread over mytomicin-C treated STO cells
as feeder cells. Every other day thereafter, the ES cell medium
containing LIF was replaced. From the 21st day after infection,
drug selection was started with addition of puromycin at a final
concentration of 1.5 .mu.g/ml. On the 28th day, the number of Nanog
GFP positive colonies (GFP, the expression of which is induced by a
Nanog gene promoter region, can be observed with the use of
fluorescent microscopy) was counted. As a control, DsRed was used
in place of miRNA. The results are shown in FIG. 1. It was found
that mmu-miR-294 and mmu-miR-295 could respectively improve the
nuclear reprogramming efficiency when introduced into mouse
embryonic fibroblasts together with three factors of Oct3/4, Sox2,
and Klf4, and could enable efficient establishment of induced
pluripotent stem cells.
Example 2
Preparation of Induced Pluripotent Stem Cells Through Nuclear
Reprogramming of Mouse Tail Tip Fibroblasts
[0109] pMXs-based retroviral vectors, which respectively encode
each of three genes of mouse-derived Oct3/4, Sox2, and Klf4, DsRed
(control), or mmu-miR-295, were transfected into PLAT-E cells using
FuGENE 6 reagent (Roche) to get retroviruses. On the next day, tail
tip fibroblasts (Nanog GFP tailtip fibroblasts) derived from
transgenic mice generated by insertion of sequences encoding EGFP
gene and puromycin resistance gene downstream of a Nanog gene
promoter region, were seeded at 1.times.10.sup.5 cells/well in
6-well plates. On the next day, these cells were infected with
retroviruses expressing three factors of Oct3/4, Sox2, and Klf4,
and either DsRed or mmu-miR-295, at a ratio of 1:1:1:1, so as to
prepare induced pluripotent stem cells through nuclear
reprogramming.
[0110] Since the third day after infection, the cells were cultured
in an ES cell medium containing LIF. On the fourth day after
infection, the cells were harvested by trypsinization and the whole
amount thereof was spread over mytomicin-C treated STO cells as
feeder cells. Every other day thereafter, the ES cell medium
containing LIF was replaced. From the 7th, 21st, or 28th day after
infection, drug selection was started with addition of puromycin at
a final concentration of 1.5 .mu.g/ml. On the 39th day, the number
of total colonies and the number of Nanog GFP positive colonies
(GFP, the expression of which is induced by Nanog promoter region,
can be observed with fluorescent microscopy) were counted. The
results are shown in FIG. 2. It was found that mmu-miR-295 could
improve the nuclear reprogramming efficiency when introduced into
mouse tail tip fibroblasts together with three factors of Oct3/4,
Sox2, and Klf4, and could accelerate the reprogramming speed and
enable efficient establishment of induced pluripotent stem
cells.
Example 3
Preparation of Induced Pluripotent Stem Cells Through Nuclear
Reprogramming of Adult Human Dermal Fibroblasts
[0111] pMXs-based retroviral vectors, which encode three genes of
human-derived OCT3/4, SOX2, and KLF4, and control DsRed or either
23 types of miRNAs or an miRNA cluster, were transfected into
PLAT-E cells using FuGENE 6 reagent (Roche) to get retroviruses. On
the next day, adult human dermal fibroblasts (aHDF) which were
generated to express a rodent ecotropic virus receptor Slc7a1
(aHDF-Slc7a1), were seeded at 3.times.10.sup.5 cells/well in 6-cm
dishes. On the next day, the cells were infected with retroviruses
expressing three genes of OCT3/4, SOX2, KLF4, and various types of
miRNAs, at a ratio of 1:1:1:1, so as to produce induced pluripotent
stem cells through nuclear reprogramming.
TABLE-US-00002 TABLE 2 miRNA sequence (other miRNA name(s)
indicated in miRBase number parentheses) accession number 1
hsa-miR-371 MI0000779 (hsa-miR-371-5p/371-3p) 2 hsa-miR-372
MI0000780 3 hsa-miR-373 MI0000781 (hsa-miR-373/373*) 4
hsa-miR-371-373 cluster 5 hsa-miR-93 MI0000095 (hsa-miR-93/93*) 6
hsa-miR-302a MI0000738 (hsa-miR-302a/302a*) 7 hsa-miR-302b
MI0000772 (hsa-miR-302b/302b*) 8 hsa-miR-302c MI0000773
(hsa-miR-302c/302c*) 9 hsa-miR-302d MI0000774 (hsa-miR-302d/302d*)
10 hsa-miR-367 MI0000775 (hsa-miR-367/367*) 11 hsa-miR-302-367
cluster 12 hsa-miR-520a MI0003149 (hsa-miR-520a-5p/520a-3p) 13
hsa-miR-520b MI0003155 14 hsa-miR-520c MI0003158
(hsa-miR-520c-5p/520c-3p) 15 hsa-miR-520d MI0003164
(hsa-miR-520d-5p/520d-3p) 16 hsa-miR-520e MI0003143 17
mmu-miR-290-295 cluster 18 mmu-miR-290 MI0000388
(mmu-miR-290-5p/290-3p) 19 mmu-miR-291a MI0000389
(mmu-miR-291a-5p/291a-3p) 20 mmu-miR-292 MI0000390
(mmu-miR-292-5p/292-3p) 21 mmu-miR-293 MI0000391 (mmu-miR-293/293*)
22 mmu-miR-294 MI0000392 (mmu-miR-294/294*) 23 mmu-miR-295
MI0000393 (mmu-miR-295/295*)
[0112] On the sixth day after infection, the cells were harvested
by trypsinization and the whole amount of 5.times.10.sup.5 cells
was spread over on mytomicin-C treated STO cells as feeder cells.
Every other day thereafter, human ES cell medium containing bFGF
(ReproCELL) was replaced. On the 24th, 32nd, and 40th day, the
number of total colonies and the number of colonies having
morphology of human ES-like cells were counted. As a control, DsRed
was used in place of miRNA. The results are shown in FIG. 3. It was
found that the number of colonies of induced pluripotent stem cells
increased twice or more, as compared to the control, by
introduction of three genes in the presence of hsa-miR-372, 373,
302b, 302-367 cluster (including 302b, 302c, 302a, 302d, and 367),
520c, mmu-miR-291a, 294, or 295.
Example 4
Preparation of Induced Pluripotent Stem Cells Through Nuclear
Reprogramming of Adult Human Dermal Fibroblasts
[0113] 3.times.10.sup.5 aHDF-Slc7a1 cells were plated on 60 mm
gelatin coated dishes and infected with retrovirus to express
DsRed, 4 factors (OCT3/4, SOX2, c-MYC, and KLF4), or 3 factors
(OCT3/4, SOX2, KLF4) in the presence of various miRNAs
independently. Six days after infection, 5.times.10.sup.5
aHDF-Slc7a1 cells were reseeded on mytomicin-C treated STO cells.
Forty days after infection, the number of human ES-like colonies
was counted. The same experiment was repeated three times.
[0114] FIG. 4A shows the results of three independent experiments.
It was found that the number of colonies of induced pluripotent
stem cells increased, as compared to the control, by introduction
of three genes in the presence of hsa-miR-372,
373/373*(hsa-miR-373), 371-373 cluster (including 371, 372, and
373), 302b/302b* (hsa-miR-302b), 302-367 cluster (including 302b,
302c, 302a, 302d, and 367), 520c-5p/520c-3p (hsa-miR-520c),
mmu-mir-290-5p/290-3p (mmu-mir-290), mmu-mir-291a-5p/291a-3p
(mmu-mir-291a), 294/294* (mmu-mir-294), or 295/295*
(mmu-mir-295).
[0115] FIG. 4B shows the morphology of ES-like colonies of iPS
cells by using microscopy.
Example 5
Expression of ES Cell Markers in iPS Cells Produced by Nuclear
Reprogramming of Mouse Tail Tip Fibroblasts (TTFs) with 4 Factors
(OCT3/4, SOX2, c-MYC, and KLF4) and with 3 Factors (OCT3/4, SOX2,
and KLF4)+mmu-miR-295/295*
[0116] 5.times.10.sup.4 FbNg TTFs (TTFs derived from Fbx15-.beta.
geo/Nanog-IRES-Puro.sup.r reporter mouse) cells were plated on
gelatin coated 6-well plates and infected with retrovirus to
express 3 factors (Oct3/4, Sox2, Klf4) plus either DsRed
(Myc(-)3f+DsRed), mmu-miR-295/295* (Myc(-)3f+mmu-miR-295/295*), or
c-Myc (4 factor). On Day 4 after infection, all the cells (Myc(
)3f+DsRed; Myc(-)3f+mmu-miR-295/295*) or 20 times diluted cells (4
factors) were reseeded on Puromycin and Hygromycin-resistant-MSTO
(PH-MSTO) cells. Puromycin selection was started on Day 7, 14, 21,
28.
[0117] RT-PCR analysis using the Rever Tra Ace Kit (Takara) showed
that the iPS cells transfected with 4 factors (OCT3/4, SOX2, c-MYC,
and KLF4), or with 3 factors (OCT3/4, SOX2, and
KLF4)+mmu-miR-295/295* expressed the ES cell specific marker genes
Oct3/4, Sox2, Nanog, and that the amounts of expression thereof
were equivalent to those obtained with mouse ES cells(ES) and mouse
iPS cells (Fbx iPS) (FIG. 5).
Example 6
Preparation of Induced Pluripotent Stem Cells Through Nuclear
Reprogramming of Mouse Embryonic Fibroblasts with 3 Factors
(Oct3/4, Klf4, and c-Myc) with miRNAs
[0118] 1.times.10.sup.5 Nanog MEFs (MEFs derived from
Nanog-IRES-Puro.sup.r reporter mouse) were plated on gelatin coated
6-well plates and infected with retrovirus to express 3 factors
(Oct3/4, c-MycWT(wild type), and Klf4) with mmu-miR-290-295
cluster, 290-5p/290-3p (mmu-miR-290), 291a-5p/291a-3p
(mmu-miR-291), 292-5p/292-3p (mmu-miR-292), 293/293* (mmu-miR-293),
294/294* (mmu-miR-294) or 295/295* (mmu-miR-295) miRNAs (1:1). On
day 4 after infection, half of the cells were reseeded on Puromycin
and Hygromycin-resistant-MSTO (PH-MSTO) cells. Puromycin selection
was started from 14 days after infection.
[0119] FIG. 6A shows the number of Nanog GFP positive colonies. The
results of three independent experiments are shown with different
colors. "DsRed" indicates the combination of Oct3/4, Klf4, c-Myc
and DsRed.
[0120] FIG. 6B shows the results of RT-PCR analysis. RT-PCR
analysis using the Rever Tra Ace Kit (Takara) showed that the iPS
cells transfected with 4 factors (OCT3/4, SOX2, c-MYC, and KLF4),
or with 3 factors (OCT3/4, SOX2, and KLF4)+mmu-miR-290-295 cluster,
291a-5p/291a-3p, 294/294* and 295/295* expressed the ES cell
specific marker genes Oct3/4, Sox2, Nanog, and that the amounts of
expression thereof were equivalent to those obtained with mouse ES
cells (ES) and mouse iPS cells (Fbx iPS).
Example 7
iPS Induction with Fb-Ng MEFs (MEFs Derived from Fbx15-.beta.
geo/Nanog-IRES-Puro.sup.r Reporter Mouse) Over-Expressing Oct3/4,
c-Myc, and Klf4 ("Sox(-)")+mmu-miR-295/295* or hsa-miR-302-367
Cluster miRNAs
[0121] 1.times.10.sup.5 Fb-Ng MEFs (MEFs derived from Fbx15-.beta.
geo/Nanog-IRES-Puro.sup.r reporter mouse) were plated on gelatin
coated 6-well plates and infected with retrovirus to express 3
factors (Oct3/4, c-MycWT(wild type), Klf4)+miR-295/295*or
hsa-miR-302-367 cluster. On day 4 after infection, cells were
reseeded on Puromycin and Hygromycin resistant mytomycin-C treated
STO cells (PH-MSTO) by in 6-well or 10 cm dishes. Puromycin
selection was started from 7 days after infection.
[0122] FIG. 7A shows cell morphology of MEFs transduced with
Oct3/4, c-Myc, and Klf4 ("Sox(-)")+mmu-miR-295/295*. The colonies
showed morphology similar to that of ES cells. FIG. 7B shows
chimeras derived from iPS cells induced with
Sox(-)3f+mmu-miR-295/295*.
[0123] FIG. 7C shows embryoid body (EB)-mediated in vitro
differentiation by human iPS cells. Human iPS cells (61B1, 61N2)
which were established by transduction of 4 genes (OCT3/4, KLF4,
SOX2, and c-MYC, i.e. "OSMK") or 3 genes (OCT3/4, KLF4, and c-MYC,
i.e., "OMK(SOX(-)")+hsa-miR-302-367 cluster miRNA were plated on a
low-binding dish, and embryoid bodies were formed on 100 mm dishes
in accordance with the method described in Takahashi et al., Cell
131:861-872, 2007. After culturing for 2 weeks, the cells were
stained using an antibody against each of .alpha.-fetoprotein
(R&D systems) which is a differentiation marker for endodermal
cells, .alpha.-smooth muscle actin (DAKO) which is a
differentiation marker for mesodermal cells, and Glial Fibrillary
Astrocytic Protein (GFAP) (DAKO) which is a differentiation marker
for ectodermal cells. The expression of each marker was confirmed
by staining. Nuclei were stained with Hoechst 33342
(Invitrogen).
Example 8
iPS Induction with 4 Factors (OCT3/4, SOX2, MYC, KLF4) or 3 Factors
(OCT3/4, MYC, KLF4, i.e., "SOX(-)3") with and without Various
miRNAs
[0124] 3.times.10.sup.5 cells of aHDF-Slc7a1 cells were plated on
60 mm gelatin coated dishes and infected with retrovirus to express
4 factors: OCT3/4, SOX2, c-MYC, KLF4 (OSMK) or 3 factors:
SOX(-)3factors (OMK) in the presence of miRNAs as indicated
(OMK:mock or miRNAs=2.5:1.5), or with 2 factors: OCT3/4+KLF4 (OK)
in the presence of miRNAs as indicated. Cells were infected with
DsRed as control. Six days after infection, 5.times.10.sup.5
aHDF-Slc7a1 cells were reseeded on mytomicin-C treated STO cells
(MSTOcells). On Day 40 after infection, the number of ES-like
colonies was counted.
[0125] TABLE 3 shows the number of human ES(hES)-like colonies in
aHDF-Slc7a1 cells transduced with OSMK, OMK with or without miRNAs,
and with OK with or without miRNAs. The hES-like colonies showed in
cells transduced with OSMK, OMK+miRNAs (hsa-miR-371-373 cluster,
hsa-miR-302-367 cluster, or hsa-miR-371-373 cluster+302-367
cluster) were detected by six independent experiments (Exp. 54, 61,
63, 114, 130, and 133).
TABLE-US-00003 TABLE 3 Number of hES-like colonies Exp. 54 Exp. 61
Exp. 63 Exp. 114 Exp. 130 Exp. 133 A control DsRed 0 0 0 0 0 0 B
OSMK Y4f (O:S:M:K = 1:1:1:1) 5 41 54 37 39 100 G Y4f (OMK:S =
2.5:1.5) 13 7 4 22 H OMK + mock Sox(-)Y3f + mock 0 0 0 0 0 0 I or
miRNA Sox(-)Y3f + h-miR-371-373 cluster 0 0 1 0 0 0 J Sox(-)Y3f +
h-miR-302-367 cluster 0 2 3 0 8 5 K Sox(-)Y3f + h-mir-371-373
cluster + 302-367 0 0 6 cluster M OK + mock OK + mock 0 0 0 N or
miRNA OK + h-miR-371-373 cluster 0 0 0 O OK + h-miR-302-367 cluster
0 0 4 P OK + h-miR-371-373 cluster + 302-367 cluster 1 0 0
[0126] FIG. 8 shows cell morphology of iPS cells induced with OSMK
(61B1); OMK (SOX(-))+hsa-miR-302-367 cluster miRNA (61N2); and
OK+hsa-miR-302-367 cluster miRNA (133O1).
INDUSTRIAL APPLICABILITY
[0127] The present invention provides an efficient method for
preparing induced pluripotent stem cells. The method of the present
invention has higher nuclear reprogramming efficiency as compared
to conventional methods. For example, safe induced pluripotent stem
cells can be efficiently produced without using c-Myc or gene
products thereof. Accordingly, the method of the present invention
enables efficient production of highly safe induced pluripotent
stem cells from a patient's own somatic cells. Cells differentiated
from such pluripotent stem cells (for example, myocardial cells,
insulin-producing cells, or nerve cells) can be safely utilized in
stem cell transplantation therapies for treatment of various
diseases, such as heart failure, insulin dependent diabetes
mellitus, Parkinson's diseases, and spinal cord injury.
[0128] Although the present invention has been described in
considerable detail with regard to certain versions thereof, other
versions are possible, and alterations, permutations, and
equivalents of the versions shown will become apparent to those
skilled in the art upon a reading of the specification and study of
the drawings. Also, the various features of the versions herein can
be combined in various ways to provide additional versions of the
present invention. Furthermore, certain terminology has been used
for the purposes of descriptive clarity, and not to limit the
present invention. Therefore, any appended claims should not be
limited to the description of the preferred versions contained
herein and should include all such alterations, permutations, and
equivalents as fall within the true spirit and scope of the present
invention.
[0129] Having now fully described this invention, it will be
understood to those of ordinary skill in the art that the methods
of the present invention can be carried out with a wide and
equivalent range of conditions, formulations, and other parameters
without departing from the scope of the invention or any
embodiments thereof.
[0130] All the disclosures of the above publication are
incorporated herein by reference.
[0131] The attached Sequence Listing includes SEQ ID NOs: 13 and
14, as well as those sequences disclosed in PCT/JP2008/59586, which
is incorporated by reference herein in its entirety.
Sequence CWU 1
1
14184RNAMus sp. 1uuccauauag ccauacucaa aauggaggcc cuaucuaagc
uuuuaagugg aaagugcuuc 60ccuuuugugu guugccaugu ggag 84269RNAMus sp.
2ggugagacuc aaaugugggg cacacuucug gacuguacau agaaagugcu acuacuuuug
60agucucucc 69367RNAHomo sapiens 3gugggccuca aauguggagc acuauucuga
uguccaagug gaaagugcug cgacauuuga 60gcgucac 67469RNAHomo sapiens
4gggauacuca aaaugggggc gcuuuccuuu uugucuguac ugggaagugc uucgauuuug
60ggguguccc 69573RNAHomo sapiens 5gcucccuuca acuuuaacau ggaagugcuu
ucugugacuu uaaaaguaag ugcuuccaug 60uuuuaguagg agu 73668RNAHomo
sapiens 6ccuuugcuuu aacauggggg uaccugcugu gugaaacaaa aguaagugcu
uccauguuuc 60aguggagg 68769RNAHomo sapiens 7ccaccacuua aacguggaug
uacuugcuuu gaaacuaaag aaguaagugc uuccauguuu 60uggugaugg
69868RNAHomo sapiens 8ccucuacuuu aacauggagg cacuugcugu gacaugacaa
aaauaagugc uuccauguuu 60gagugugg 68968RNAHomo sapiens 9ccauuacugu
ugcuaauaug caacucuguu gaauauaaau uggaauugca cuuuagcaau 60ggugaugg
681087RNAHomo sapiens 10ucucaggcug ucguccucua gagggaagca cuuucuguug
ucugaaagaa aagaaagugc 60uuccuuuuag aggguuaccg uuugaga 871182RNAMus
sp. 11ccuauguagc ggccaucaaa guggaggccc ucucuugagc cugaaugaga
aagugcuucc 60acuuugugug ccacugcaug gg 8212820RNAHomo sapiens
12guuuucuuuc uccucagcuc uaaauacucu gaaguccaaa gaaguuguau guuggguggg
60cucccuucaa cuuuaacaug gaagugcuuu cugugacuuu aaaaguaagu gcuuccaugu
120uuuaguagga gugaauccaa uuuacuucuc caaaauagaa cacgcuaacc
ucauuugaag 180ggauccccuu ugcuuuaaca uggggguacc ugcuguguga
aacaaaagua agugcuucca 240uguuucagug gaggugucuc caagccagca
caccuuuugu uacaaaauuu uuuuguuauu 300guguuuuaag guuacuaagc
uuguuacagg uuaaaggauu cuaacuuuuu ccaagacugg 360gcuccccacc
acuuaaacgu ggauguacuu gcuuugaaac uaaagaagua agugcuucca
420uguuuuggug augguaaguc uuccuuuuac auuuuuauua uuuuuuuaga
aaauaacuuu 480auuguauuga ccgcagcuca uauauuuaag cuuuauuuug
uauuuuuaca ucuguuaagg 540ggcccccucu acuuuaacau ggaggcacuu
gcugugacau gacaaaaaua agugcuucca 600uguuugagug uggugguucc
uaccuaauca gcaauugcgu uaacgcccac acugugugca 660guucuuggcu
acaggccauu acuguugcua auaugcaacu cuguugaaua uaaauuggaa
720uugcacuuua gcaaugguga uggauuguua agccaaugac agaauuuaaa
ccacagacuu 780acuuugauag cacucuuaau gguauaacuu cuucucccau
82013965DNAHomo sapiens 13cttcaacagc tcatcaaggg ctactctcca
cctccttgct taaaagcctc ttctgatggg 60taagtgcttc cacttgcgat cgccgccttg
ccgcatcccc tcagcctgtg gcactcaaac 120tgtgggggca ctttctgctc
tctggtgaaa gtgccgccat cttttgagtg ttaccgcttg 180agaagactca
acctgcggag aagataccat tttgattggg tgagggggcg ggtagcagga
240tggccctaga ccctgcctat ggccgtttcc tcgtgatata aatttcttgg
ccggggctct 300tgcagatgga gctgctcacc ctgtgggcct caaatgtgga
gcactattct gatgtccaag 360tggaaagtgc tgcgacattt gagcgtcacc
ggtgacgccc atatcaacgg atgccgtgga 420gctcggtctt ctgcaggaac
taaagagcct gtggtttcga ttcccagccg gaaactgtct 480tgggtacagg
tcccttacag cgtctggctg taatggctcc ggaaaacctg gggaagggaa
540gggggcctcc tgggctctca cctgacacaa ctaagggaat ctgggttaga
tggtgaaagg 600aagagaaggt tcagagggcg gctgtgcgcc tgcgccgggc
gcggcggctc acacctgtaa 660tcccagcacg ttgggaggcc gaggcaggcg
gatcacgagg tcaggagatc gagaccatcc 720tggctaacac ggtgaaaccc
cgtctctact aaaaatacaa aaaatgagcc gggcgtggtg 780gcgggcgcct
gtagtcccag ctacttggga ggctgaggca ggagaatggc gtgaacccgg
840gaggcggagt ttgcagtgag ccgagatcgc gccaccgcac tccagcctgg
gcgacagagc 900aagactcatt ctcaaagaaa aaaaaaaaaa aaaacagcag
ctgtgaccaa ggggctgtat 960gcaca 9651483DNAMus sp. 14ctcatcttgc
ggtactcaaa ctatgggggc actttttttt ttctttaaaa agtgccgcct 60agttttaagc
cccgccggtt gag 83
* * * * *
References