U.S. patent application number 12/733118 was filed with the patent office on 2010-11-04 for method of nuclear reprogramming.
Invention is credited to Keisuke Okita, Shinya Yamanaka.
Application Number | 20100279404 12/733118 |
Document ID | / |
Family ID | 41255176 |
Filed Date | 2010-11-04 |
United States Patent
Application |
20100279404 |
Kind Code |
A1 |
Yamanaka; Shinya ; et
al. |
November 4, 2010 |
METHOD OF NUCLEAR REPROGRAMMING
Abstract
This invention provides a method of producing an induced
pluripotent stem cell comprising the step of introducing at least
one kind of non-viral expression vector (more preferably a plasmid
vector) incorporating at least one gene that encodes a
reprogramming factor into a somatic cell. An induced pluripotent
stem cell wherein no exogenous genes induced is integrated into the
cellular genome is also provided.
Inventors: |
Yamanaka; Shinya; (Kyoto,
JP) ; Okita; Keisuke; (Kyoto, JP) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET, FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
41255176 |
Appl. No.: |
12/733118 |
Filed: |
May 1, 2009 |
PCT Filed: |
May 1, 2009 |
PCT NO: |
PCT/JP2009/058873 |
371 Date: |
May 3, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61071508 |
May 2, 2008 |
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61136246 |
Aug 21, 2008 |
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61136615 |
Sep 19, 2008 |
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61193636 |
Dec 11, 2008 |
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Current U.S.
Class: |
435/366 ;
435/320.1; 435/325; 435/377 |
Current CPC
Class: |
C12N 2506/1361 20130101;
C12N 2501/602 20130101; C12N 2501/603 20130101; C12N 2501/605
20130101; C12N 2501/608 20130101; C12N 15/79 20130101; C12N 2506/13
20130101; C12N 2510/00 20130101; C12N 5/0696 20130101; C12N
2506/1307 20130101; C12N 2506/02 20130101; C12N 2501/604 20130101;
C12N 2501/606 20130101 |
Class at
Publication: |
435/366 ;
435/377; 435/325; 435/320.1 |
International
Class: |
C12N 5/0735 20100101
C12N005/0735; C12N 15/79 20060101 C12N015/79 |
Claims
1. A method of producing an induced pluripotent stem cell,
comprising the step of introducing at least one kind of non-viral
expression vector incorporating at least one gene that encodes a
reprogramming factor into a somatic cell.
2. The method of claim 1, wherein the vector is a non-viral
expression vector autonomously replicable outside a chromosome.
3. The method of claim 1, wherein the vector is a plasmid
vector.
4. (canceled)
5. The method of claim 1, wherein the gene that encodes a
reprogramming factor is one or more kind of genes selected from the
group consisting of an Oct family gene, a Klf family gene, a Sox
family gene, a Myc family gene, a Lin family gene, and the Nanog
gene.
6. The method of claim 1, wherein the gene that encodes a
reprogramming factor is one of the following combinations: (a) a
combination of two kinds of genes consisting of an Oct family gene
and a Sox family gene; (b) a combination of three kinds of genes
consisting of an Oct family gene, a Klf family gene, and a Sox
family gene; (c) a combination of four kinds of genes consisting of
an Oct family gene, a Klf family gene, a Sox family gene, and a Myc
family gene; (d) a combination of four kinds of genes consisting of
an Oct family gene, a Sox family gene, a Lin family gene, and the
Nanog gene; and (e) a combination of six kinds of genes consisting
of an Oct family gene, a Sox family gene, a Klf family gene, a Myc
family gene, a Lin family gene, and the Nanog gene, or any one of
these combinations further comprising the TERT gene and/or the SV40
Large T antigen gene.
7. The method of claim 1, wherein the gene that encodes a
reprogramming factor is one of the following combinations: (a) a
combination of two kinds of genes consisting of Oct3/4 and Sox2;
(b) a combination of three kinds of genes consisting of Oct3/4,
Klf4, and Sox2; (c) a combination of four kinds of genes consisting
of Oct3/4, Klf4, Sox2, and c-Myc; (d) a combination of four kinds
of genes consisting of Oct3/4, Sox2, Lin28, and Nanog; and (e) a
combination of six kinds of genes consisting of Oct3/4, Sox2, Klf4,
c-Myc, Lin28, and Nanog, or any one of these combinations further
comprising the TERT gene and/or the SV40 Large T antigen gene.
8. The method of claim 1, wherein the number of kinds of non-viral
expression vectors introduced into the somatic cell is 1, 2, 3, or
4.
9. The method of claim 8, wherein the gene that encodes a
reprogramming factor is a combination of three kinds of genes
consisting of an Oct family gene, a Klf family gene, and a Sox
family gene, or a combination of four kinds of genes consisting of
an Oct family gene, a Klf family gene, a Sox family gene, and a Myc
family gene, wherein the Oct family gene, the Klf family gene, and
the Sox family gene are incorporated in one kind of non-viral
expression vector.
10. The method of claim 9, wherein the Oct family gene, the Klf
family gene, and the Sox family gene are incorporated in one kind
of non-viral expression vector in this order in the orientation
from the 5' to 3' end.
11. The method of claim 9, wherein the Oct family gene, the Klf
family gene, and the Sox family gene are incorporated in one kind
of non-viral expression vector with an intervening sequence
enabling polycistronic expression.
12. The method of claim 9, wherein the Oct family gene is Oct3/4,
the Klf family gene is Klf4, the Sox family gene is Sox2, and the
Myc family gene is c-Myc.
13. The method of claim 9, wherein a first non-viral expression
vector and a second non-viral expression vector are concurrently
introduced into a somatic cell.
14. The method of claim 13, wherein said introduction is repeatedly
performed twice or more.
15. The method of claim 1, wherein the somatic cell is a somatic
cell of a mammal, including a human.
16. An induced pluripotent stem cell that can be obtained by the
method of claim 1.
17. The induced pluripotent stem cell of claim 16, wherein whole or
part of the at least one non-viral expression vector introduced is
substantially not integrated in the chromosome.
18. A non-viral expression vector incorporating a gene that encodes
at least one reprogramming factor.
19. The vector of claim 18, wherein the vector is a plasmid
vector.
20. The vector of claim 18, wherein an Oct family gene, a Klf
family gene, and a Sox family gene are incorporated.
21. The vector of claim 20, wherein the Oct family gene, the Klf
family gene, and the Sox family gene are incorporated in this order
in the orientation from the 5' to 3' end.
22. The vector of claim 21, wherein the Oct family gene, the Klf
family gene, and the Sox family gene are incorporated via a
sequence enabling polycistronic expression.
23. The vector of claim 20, wherein the Oct family gene is Oct3/4,
the Klf family gene is Klf4, and the Sox family gene is Sox2.
24. An induced pluripotent stem cell wherein one or more transgenes
encoding reprogramming factor are integrated into the cellular
genome in the form of plasmid.
25. An induced pluripotent stem cell wherein no exogenous genes
introduced is integrated into the cellular genome.
26. The vector of claim 18, harboring genes encoding two or more
kinds of reprogramming factors, wherein the genes are incorporated
via 2A sequence derived from foot-and-mouth disease virus.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method of reprogramming a
somatic cell and producing an induced pluripotent stem cell.
BACKGROUND ART
[0002] Established from human or mouse early embryos, embryonic
stem cells (ES cells) are capable of being cultured for a long time
while maintaining their potential for differentiating into all
types of cells found in a living organism. With this feature, human
ES cells are expected to serve for cell transplantation therapies
for many diseases, including Parkinson's disease, juvenile
diabetes, and leukemia. However, ES cell transplantation,poses the
problem of causing rejections as with organ transplantation.
Additionally, not a few people oppose the use of ES cells
established with the destruction of a human embryo, from an ethical
viewpoint.
[0003] If the dedifferentiation of a patient's somatic cells is
induced to establish cells possessing pluripotency and
proliferating capability similar to those of an ES cell (herein
these cells are referred to as "induced pluripotent stem cells"
(iPS cells), and sometimes referred to as "embryonic stem cell-like
cells" or "ES-like cells"), the established cells will be useful as
ideal pluripotent cells that do not pose the problems of rejections
and ethical issues. In recent years, it has been reported that iPS
cells can be produced from mouse and human differentiated cells,
arousing great attention (International Patent Application
Publication No. WO2007/69666; Cell, 126, pp. 663-676, 2006; Cell,
131, pp. 861-872, 2007; Science, 318, pp. 1917-1920, 2007; Nature,
451, pp. 141-146, 2008).
[0004] All these methods comprise the step of introducing a
plurality of particular nuclear reprogramming factors (e.g., in
Cell, 126, pp. 1-14, 2006, 4 factors are used: Oct3/4, Sox2, Klf4,
and c-Myc) into a somatic cell to achieve reprogramming, which step
involves the use of a retrovirus or a lentivirus for the purpose of
introducing the genes that encode the nuclear reprogramming factors
into a somatic cell efficiently. However, since gene delivery using
a viral vector involves safety issues, there is a demand for
developing a method of producing iPS cells without using a viral
vector.
SUMMARY OF THE INVENTION
Technical Problem
[0005] It is an object of the present invention to provide a method
of producing an iPS cell by reprogramming a somatic cell without
using a viral vector such as a retrovirus.
Solution to Problem
[0006] The present inventors extensively investigated to solve the
problems described above, and found that an iPS cell can be
produced by introducing genes that encode reprogramming factors
into a somatic cell by means of a non-viral expression vector such
as a plasmid vector, and that a safe iPS cell can be obtained from
a somatic cell by the method. The present invention has been
developed on the basis of these findings.
[0007] Accordingly, the present invention provides a method of
producing an induced pluripotent stem cell, comprising the step of
introducing at least one kind of non-viral expression vector
incorporating at least one gene that encodes a reprogramming factor
into a somatic cell.
[0008] In a preferred embodiment, the present invention provides
the above-described method wherein the vectors are non-viral
expression vectors autonomously replicable outside a chromosome;
and the above-described method wherein the vector is a plasmid
vector.
[0009] In another preferred embodiment, the present invention
provides the above-described method wherein the gene that encodes a
reprogramming factor is one of genes selected by a method of
screening for nuclear reprogramming factors described in WO
2005/80598 or a combination of a plurality of such genes; and the
above-described method wherein the gene that encodes a
reprogramming factor is one or more kinds of genes selected from
the group consisting of an Oct family gene, a Klf family gene, a
Sox family gene, a Myc family gene, a Lin family gene, and the
Nanog gene, preferably a combination of two kinds of genes, more
preferably a combination of three kinds of genes, particularly
preferably a combination of four or more kinds of genes.
[0010] More preferable combinations are (a) a combination of two
kinds of genes consisting of an Oct family gene and a Sox family
gene; (b) a combination of three kinds of genes consisting of an
Oct family gene, a Klf family gene, and a Sox family gene; (c) a
combination of four kinds of genes consisting of an Oct family
gene, a Klf family gene, a Sox family gene, and a Myc family gene;
(d) a combination of four kinds of genes consisting of an Oct
family gene, a Sox family gene, a Lin family gene, and the Nanog
gene; (e) a combination of six kinds of genes consisting of an Oct
family gene, a Klf family gene, a Sox family gene, a Myc family
gene, a Lin family gene, and the Nanog gene;and the like.
Furthermore, it is also preferable to include the TERT gene and/or
the SV40 Large T antigen gene in the combination. As the case may
be, it is preferable to exclude Klf family genes.
[0011] Particularly preferred combinations thereof are a
combination of two kinds of genes consisting of Oct3/4 and Sox2; a
combination of three kinds of genes consisting of Oct3/4, Klf4, and
Sox2; a combination of four kinds of genes consisting of Oct3/4,
Klf4, Sox2, and c-Myc; a combination of four kinds of genes
consisting of Oct3/4, Sox2, Lin28, and Nanog; and a combination of
six kinds of genes consisting of Oct3/4, Klf4, Sox2, c-Myc, Lin28,
and Nanog. It is also preferable to include the TERT gene and/or
the SV40 Large T antigen gene in these combinations. As the case
may be, it is preferable to exclude Klf4.
[0012] In another preferred embodiment, the present invention
provides the above-described method wherein the number of kinds of
non-viral expression vectors introduced into a somatic cell is 1,
2, 3, or 4; the above-described method wherein the genes that
encode reprogramming factors are a combination of three kinds of
genes consisting of an Oct family gene, a Klf family gene, and a
Sox family gene, and these genes are incorporated in one kind of
non-viral expression vector; the above-described method wherein the
genes that encode nuclear reprogramming factors are a combination
of four kinds of genes consisting of an Oct family gene, a Klf
family gene, a Sox family gene, and a Myc family gene, and the Oct
family gene, the Klf family gene, and the Sox family gene are
incorporated in one kind of non-viral expression vector; the
above-described method wherein the Oct family gene, the Klf family
gene, and the Sox family gene are incorporated in one kind of
non-viral expression vector in this order in the orientation from
the 5' to 3' end; and the above-described method wherein the Oct
family gene, the Klf family gene, and the Sox family gene are
incorporated in one kind of non-viral expression vector with an
intervening sequence enabling polycistronic expression.
[0013] In another preferred embodiment, the present invention
provides the above-described method wherein two or more kinds of
the above-described non-viral expression vectors are concurrently
introduced into a somatic cell; the above-described method wherein
the genes that encode reprogramming factors are a combination of
four kinds of genes consisting of an Oct family gene, a Klf family
gene, a Sox family gene, and a Myc family gene, and a first
non-viral expression vector incorporating three or less kinds of
genes selected from among the four kinds of genes, and a second
non-viral expression vector incorporating the remaining gene(s) out
of the four kinds of genes are concurrently introduced into a
somatic cell; the above-described method wherein the three or less
kinds of genes are an Oct family gene, a Klf family gene, and a Sox
family gene, and the remaining gene is a Myc family gene; the
above-described method wherein the three or less kinds of genes are
Oct3/4, Klf4, and Sox2, and the remaining gene is c-Myc; and the
above-described method wherein introduction of the non-viral
expression vector into a somatic cell is repeatedly performed twice
or more.
[0014] In a particularly preferred embodiment, the present
invention provides the above-described method wherein a first
non-viral expression vector harboring Oct3/4, Klf4, and Sox2, and a
second non-viral expression vector harboring c-Myc are introduced
into a somatic cell; the above-described method wherein a first
non-viral expression vector harboring Oct3/4, Klf4, and Sox2 in
this order in the orientation from the 5' to 3' end, and a second
non-viral expression vector harboring c-Myc are introduced into a
somatic cell; the above-described method wherein Oct3/4, Klf4, and
Sox2 are ligated in this order in the orientation from the 5' to 3'
end with an intervening sequence enabling polycistronic expression
and inserted into the first non-viral expression vector; the
above-described method wherein the first non-viral expression
vector and the second non-viral expression vector are concurrently
introduced into a somatic cell; and the above-described method
wherein the introduction is repeatedly performed twice or more.
Also provided is the above-described method wherein whole or prat
of the at least one non-viral expression vector introduced is
substantially not integrated in the chromosome.
[0015] In another preferred embodiment, the present invention
provides the above-described method wherein the somatic cell is a
somatic cell of a mammal, including a human, preferably a human or
mouse somatic cell, particularly preferably a human somatic cell;
the above-described method wherein the somatic cell is a fetal
human cell or a somatic cell derived from an adult human; and the
above-described method wherein the somatic cell is a somatic cell
collected from a patient.
[0016] In another aspect, the present invention provides an induced
pluripotent stem cell that can be obtained by the above-described
method. In a preferred embodiment, the present invention also
provides an induced pluripotent stem cell wherein all or some of
the at least one non-viral expression vector introduced is
substantially not integrated in the chromosome.
[0017] Also provided are the above-described induced pluripotent
stem cell wherein the somatic cell is a somatic cell of a mammal,
including a human, preferably a human or mouse somatic cell,
particularly preferably a human somatic cell; the above-described
induced pluripotent stem cell wherein the somatic cell is a fetal
human cell or a somatic cell derived from an adult human; and the
above-described induced pluripotent stem cell wherein the somatic
cell is a somatic cell collected from a patient.
[0018] A non-viral expression vector, preferably a plasmid vector,
for use in the above-described method of producing an induced
pluripotent stem cell, incorporating at least one gene that encodes
a reprogramming factor, is also provided by the present
invention.
[0019] A somatic cell induced and differentiated from the
above-described induced pluripotent stem cell is also provided by
the present invention.
[0020] The present invention also provides a stem cell therapy
comprising the step of transplanting to a patient a somatic cell
obtained by differentiation induction of an induced pluripotent
stem cell obtained by the above-described method using a somatic
cell separated from the patient.
[0021] The present invention further provides a method of
evaluating the physiological activities and toxicities of
compounds, drugs, poisonous substances and the like using various
cells obtained by differentiation induction of an induced
pluripotent stem cell obtained by the above-described method.
Advantageous Effects of Invention
[0022] Produced without using a vector to be integrated into a
chromosome, such as a retrovirus, the induced pluripotent stem cell
provided by the present invention is advantageous in that
tumorigenesis and other problems do not arise in the somatic cells
and tissues obtained by differentiating the induced pluripotent
stem cell. In a preferred embodiment of the present invention, in
the induced pluripotent stem cell produced by the method of the
present invention, all or some of the at least one non-viral
expression vector introduced is episomally present, substantially
not integrated in the chromosome. Therefore, the method of the
present invention makes it possible to prepare a highly safe
induced pluripotent stem cell from, for example, a patient's
somatic cell, and the cells obtained by differentiating this cell
(e.g., myocardial cells, insulin-producing cells, or nerve cells
and the like) can be safely used for stem cell transplantation
therapies for a broad range of diseases, including heart failure,
insulin-dependent diabetes, Parkinson's disease and spinal
injury.
BRIEF DESCRIPTION OF DRAWINGS
[0023] FIG. 1 shows a time course protocol for transfecting a
somatic cell (MEF) with Oct3/4, Klf4, Sox2, and c-Myc using
plasmids according to the method of the present invention, results
of seven independent tests (left photographs, 432A-1 to 432A-7:
cell density 1.times.10.sup.6 cells/100 mm dish) and results of
another test (right photographs, 432B-1: cell density
2.times.10.sup.5 cells/100 mm dish). The lowermost panels in the
center show control results (no transfection). In FIG. 1, the Phase
columns show phase-contrast images, and the GFP columns show
GFP-positive colonies.
[0024] FIG. 2 shows an expression plasmid for iPS cell production.
Three kinds of cDNAs that encode Oct3/4, Klf4, and Sox2 were
ligated in this order with sequence encoding the 2A peptide as
intervening sequence, and inserted into the pCX plasmid
(pCX-2A-mOKS). Furthermore, a cDNA of c-Myc was inserted into pCX
(pCX-c-Myc). The bald lines show the amplification regions used in
the PCR analysis for detecting plasmid integration in the genome
(FIG. 6).
[0025] FIG. 3 shows the time schedules for iPS cell induction using
plasmids. The solid arrows indicate the time points of transfection
of the respective plasmids.
[0026] FIG. 4 shows the morphology of non-virus mediated iPS cells
established. The upper panels show phase-contrast images, and the
lower panels show GFP-positive colonies (scale bar=200 .mu.m).
[0027] FIG. 5 shows results of PCR analysis for the genetic
expression of ES cell markers, obtained using total RNAs isolated
from ES cells, IFS cells induced using retroviruses (clone 20D-17:
Nature, 448, pp. 313-317, 2007), iPS cells induced using plasmids
(clones 440A-3, 4, 7, 8, 10 and 11; clone 432A-1), and MEF
cells.
[0028] FIG. 6 shows the detection of plasmid integration by PCR.
Genomic DNAs were extracted from a C57BL/6 mouse, iPS cell induced
using retroviruses (clone 20D-17), iPS cells induced with plasmids
(clone 432A-1; clones 440A-1 to 11) and MEF cells, and analyzed by
PCR using the primers shown in FIGS. 2, 13 and 14. In the PCR for
O-1, K and M, the bands derived from endogenous genes are indicated
by the outlined arrowheads, and the bands derived from integrated
plasmids are indicated by the solid arrowheads. For the Fbx15
reporter, the lower band indicates wild-type alleles, and the upper
band indicates knocked-in alleles.
[0029] FIG. 7 shows results of teratoma formation. iPS cells
without integration of plasmids (clones 440A-3, -4, and -8) were
subcutaneously transplanted to nude mice. Four weeks later, tumors
were resected and stained with hematoxylin and eosin. Shown from
above are the results for gut-like epithelial tissue, epidermal
tissue, striated muscles, and nerve tissue, respectively (scale
bar=50 .mu.m).
[0030] FIG. 8 shows chimeric mice derived from iPS cells without
integration (clones 440A-3 and -8).
[0031] FIG. 9 shows the detection of integration of plasmids by
PCR. Genomic DNAs were extracted from an ICR mouse, iPS cell (clone
432A-1), and chimeric mice derived from iPS cells induced using
plasmids (clone 432A-1; clones 440A-3, 8), and the O-1, K and M
regions shown in FIG. 2 were amplified by PCR. The bands derived
from endogenous genes are indicated by the outlined arrowheads, and
the bands derived from integrated plasmids are indicated by the
solid arrowheads. The presence of the Nanog reporter and Fbx15
reporter was also detected by PCR.
[0032] FIG. 10 shows the probes used in Southern blot analysis and
the positions of the restriction endonuclease recognition sites. E
indicates EcoRI, and B indicates BamHI.
[0033] FIG. 11 shows results of Southern blot analysis. Genomic
DNAs (6 .mu.g) were extracted from RF8 ES cells and iPS cells
(clones 440A-3, 4, 7, 8, 10, and 11; clone 432A-1), and cleaved
with BamHI and EcoRI. A mixture of the pCX-2A-mOKS and pCX-c-Myc
plasmids (each 20 pg) served for control. The outlined arrowheads
indicate the bands derived from endogenous genes, and the solid
arrowhead indicates the band derived from the Oct3/4 pseudogene
(estimated size 2049 bp) on chromosome 3. The arrows indicate the
bands derived from transgenes. Although the identities of the many
bands observed in clone 432A-1 are unclear, this may suggest the
integration of multiple transgenes. The GFP probe was used to
detect Nanog reporter alleles.
[0034] FIG. 12 shows results of SSLP analysis. On genomic DNAs
(each 50 ng) from C57BL/6 mouse, RF8 ES cell, iPS cells without
integration (clones 440A-3 to 11) and MEF cells, SSLP analysis was
performed. These iPS cells derive from a mixture of five MEF cell
clones (clones 1, 2, 3, 5, and 6).
[0035] FIGS. 13 and 14 show the primers used for PCR in Examples 1
to 3.
[0036] FIG. 15 shows a time course protocol for transfecting human
dental pulp stem cells with Oct3/4, Klf4, Sox2, c-Myc, Lin28, Nanog
and the SV40 Large T antigen using plasmids according to the method
of the present invention, and 16 independent iPS cell colonies.
[0037] FIGS. 16 and 17 show photographs of iPS cells established
from fetal HDF (5 clones: 203A-1 to 203A-5, of which 203A-4 was
picked up as a negative control) on day 31 after transfection (FIG.
16) and in the 2nd subculture (FIG. 17).
[0038] FIG. 18 shows the results of genomic-PCR analysis of 5 iPS
cell clones (203A-1 to 203A-5).
[0039] FIGS. 19 and 20 show photographs of iPS cells established
from human dental pulp stem cells (5 clones: 217A-1 to -4 and -6)
on day 35 after transfection (FIG. 19) and in the 2nd subculture
(FIG. 20).
[0040] FIG. 21 shows the results of genomic-PCR analysis of 5 iPS
cell clones (217A-1 to -4 and -6).
[0041] FIGS. 22 and 23 show photographs of IFS cells established
from young female HDF (2 clones: 279A-1 and -2) on day 35 after the
first electroporation (FIG. 22) and clone 279A-2 after passage
culture (FIG. 23; the right panel is a closeup picture of the boxed
area in the left panel).
[0042] FIG. 24 shows the results of genomic-PCR analysis of iPS
cell clone 279A-2 demonstrating the integration of the
transgenes.
[0043] FIG. 25 shows photographs of iPS cells (8 clones: 497A-1 to
A-8) after the selection (colonies were selected on day 25 after
transfection). The upper panels show phase-contrast images, and the
lower panels show GFP-positive colonies.
[0044] FIG. 26 shows the results of genomic-PCR analysis of 5 iPS
cell clones (497A-1 to A-5). In 497A-2 and 497A-5, no exogenous
gene was not integrated into the genome.
DESCRIPTION OF EMBODIMENTS
[0045] The method of the present invention is intended to produce
an induced pluripotent stem cell, comprising the step of
introducing at least one kind of non-viral expression vector
incorporating at least one gene that encodes a reprogramming factor
into a somatic cell. The non-viral expression vector is preferably
an expression vector autonomously replicable outside a chromosome,
more preferably a plasmid expression vector.
[0046] As an example of a means for identifying a nuclear
reprogramming factor, a nuclear reprogramming factor screening
method described in WO 2005/80598 can be utilized. All disclosures
therein are incorporated herein by reference. Those skilled in the
art are able to screen for nuclear reprogramming factors, and to
utilize them for the method of the present invention, by referring
to the aforementioned publication. It is also possible to identify
nuclear reprogramming factors using a method modified or altered
from the above-described screening method.
[0047] Some examples of combinations of genes that encode
reprogramming factors are disclosed in WO2007/69666. All
disclosures therein are incorporated herein by reference. Those
skilled in the art are able to choose genes that can suitably be
used in the method of the present invention as appropriate by
referring to the aforementioned publication. Other examples of
combinations of genes that encode reprogramming factors are given
in Science, 318, pp. 1917-1920, 2007, WO2008/118820 and the like.
Therefore, those skilled in the art are able to understand the
diversity of combinations of genes that encode nuclear
reprogramming factors; by utilizing a nuclear reprogramming factor
screening method described in WO 2005/80598, appropriate
combinations of genes other than the combinations described in
WO2007/69666 and Science, 2007 (supra) can be utilized in the
method of the present invention.
[0048] Preferable genes that encode reprogramming factors include
one or more kind of genes selected from the group consisting of an
Oct family gene, a Klf family gene, a Sox family gene, a Myc family
gene, a Lin family gene, and the Nanog gene, preferably a
combination of two kinds of genes, more preferably of three kinds
of genes, and particularly preferably of four kinds of genes.
[0049] Examples of Oct family genes, Klf family genes, Sox family
genes, and Myc family genes are given in WO2007/69666. Likewise,
for Lin family genes, those skilled in the art are likewise able to
extract a family gene. For example, as examples of Lin family
genes, Lin28 and Lin28B may be included.
[0050] More preferable combinations include, but are not limited
to, [0051] (a) a combination of two kinds of genes consisting of an
Oct family gene and a Sox family gene; [0052] (b) a combination of
three kinds of genes consisting of an Oct family gene, a Klf family
gene, and a Sox family gene; [0053] (c) a combination of four kinds
of genes consisting of an Oct family gene, a Klf family gene, a Sox
family gene, and a Myc family gene; [0054] (d) a combination of
four kinds of genes consisting of an Oct family gene, a Sox family
gene, a Lin family gene, and the Nanog gene; [0055] (e) a
combination of six kinds of genes consisting of an Oct family gene,
a Sox family gene, a Klf family gene, a Myc family gene, a Lin
family gene, and the Nanog gene; and the like.
[0056] All these genes are present in common in mammals, including
humans. Genes derived from optionally chosen mammals (e.g., humans,
mice, rats, bovines, sheep, horses, monkeys) can be used in the
present invention. In addition to wild-type gene, mutant genes
whose translation products have several (e.g., 1 to 10, preferably
1 to 6, more preferably 1 to 4, more preferably 1 to 3,
particularly preferably 1 or 2) amino acids substituted, inserted,
and/or deleted, and possess a function similar to that of the wild
type gene product, can also be utilized. For example, as c-Myc
genes, the wild type, a gene encoding stable type mutant (T58A) and
the like may be used. The same applies to other gene products.
[0057] In addition to the aforementioned genes, a gene that encodes
a factor that induces cell immortalization may further be combined.
As disclosed in WO2007/69666, for example, the TERT gene, and one
or more kind of genes selected from the group consisting of the
following genes: SV40 Large T antigen, HPV16 E6, HPV16 E7, and
Bmil, can be used singly, or in combination as appropriate.
[0058] Examples of preferable combinations include: [0059] (f) a
combination of five kinds of genes consisting of an Oct family
gene, a Klf family gene, a Sox family gene, a Myc family gene, and
the TERT gene; [0060] (g) a combination of five kinds of genes
consisting of an Oct family gene, a Klf family gene, a Sox family
gene, a Myc family gene, and the SV40 Large T antigen gene; [0061]
(h) a combination of six kinds of genes consisting of an Oct family
gene, a Klf family gene, a Sox family gene, a Myc family s gene,
the TERT gene, and the SV40 Large T antigen gene; and [0062] (i) a
combination of seven kinds of genes consisting of an Oct family
gene, a Klf family gene, a Sox family gene, a Myc family gene, a
Lin family gene, the Nanog gene, and the TERT gene or the SV40
Large T antigen gene.
[0063] As required, the Klf family gene may be excluded from the
aforementioned combinations.
[0064] Furthermore, in addition to the aforementioned genes, one or
more kind of genes selected from the group consisting of Fbx15,
ERas, ECAT15-2, Tcl1, and .beta.-catenin may be combined, and/or
one or more kind of genes selected from the group consisting of
ECAT1, Esg1, Dnmt3L, ECAT8, Gdf3, Sox15, ECAT15-1, Fth117, Sa114,
Rex1, UTF1, Stella, Stat3, and Grb2 may also be combined. These
combinations are specifically described in WO2007/69666.
[0065] If one or more kind of these genes are already expressed in
the somatic cell to be reprogrammed, the gene(s) can be excluded
from the genes to be introduced. When one or more kind of these
genes are introduced into a somatic cell to be reprogrammed using a
vector to be integrated into a chromosome, such as a retrovirus,
the remaining one or more genes can be introduced using a non-viral
expression vector according to the method of the present invention.
Alternatively, when one or more kind of the gene products of these
genes are introduced into a nucleus by means of fused protein or
nuclear microinjection, the remaining one or more genes can be
introduced using a non-viral expression vector according to the
method of the present invention.
[0066] Particularly preferable combinations of genes are, [0067]
(1) a combination of two kinds of genes consisting of Oct3/4 and
Sox2; [0068] (2) a combination of three kinds of genes consisting
of Oct3/4, Klf4, and Sox2; [0069] (3) a combination of four kinds
of genes consisting of Oct3/4, Klf4, Sox2, and c-Myc; [0070] (4) a
combination of four kinds of genes consisting of Oct3/4, Sox2,
Lin28, and Nanog; [0071] (5) a combination of five kinds of genes
consisting of Oct3/4, Sox2, c-Myc, TERT, and SV40 Large T antigen;
[0072] (6) a combination of six kinds of genes consisting of
Oct3/4, Klf4, Sox2, c-Myc, TERT, and SV40 Large T antigen; [0073]
(7) a combination of six kinds of genes consisting of Oct3/4, Klf4,
c-Myc, Sox2, Lin28, and Nanog; [0074] (8) a combination of seven
kinds of genes consisting of Oct3/4, Klf4, c-Myc, Sox2, Lin28,
Nanog, and TERT or SV40 Large T antigen, [0075] and the like.
[0076] In addition to the aforementioned genes, a gene that encodes
a factor that induces cell immortalization may further be combined.
As disclosed in WO2007/69666, for example, one kind or more of
genes selected from the group consisting of the TERT gene, and the
following genes: HPV16 E6, HPV16 E7, and Bmil, can be used singly,
or in combination as appropriate.
[0077] When reprogramming is performed using nerve stem cells
endogenously expressing Sox2 and c-Myc, or the like as a somatic
cell source, a combination of two kinds of genes consisting of
Oct3/4 and Klf4, or a combination of two kinds of genes consisting
of Oct3/4 and c-Myc (see Nature, Published online, 29 Jun. 2008,
p1-5 (doi:10.1038/nature07061)) can also be mentioned.
[0078] In the combinations (3), (5), (6), and (7) above, L-Myc can
be used in place of c-Myc.
[0079] It should be noted that combinations of genes are not
limited thereto. Additionally, the scope of the present invention
includes a method wherein one or more genes selected from among the
above-described genes are introduced into a somatic cell using a
non-viral expression vector, and the remaining gene or gene product
is introduced into the somatic cell by another means. For example,
it is also possible to introduce one or more genes selected from
among the above-described genes into a somatic cell using a
non-viral expression vector, and to introduce the remaining gene
into the somatic cell using a viral vector such as retroviral
vector, lentiviral vector, adenoviral vector, adeno-associated
viral vector, Sendai viral vector.
[0080] When two or more kinds of genes that encode reprogramming
factors are introduced into a somatic cell using non-viral
expression vectors, some of the two or more kinds of genes to be
introduced can be introduced into a somatic cell at a time
different from that for other genes, or all kinds of genes to be
introduced can be concurrently introduced into a somatic cell;
however, it is preferable that all genes to be introduced be
concurrently introduced into a somatic cell. When two or more kinds
of different non-viral expression vectors are used to introduce two
or more kinds of genes, all kinds of non-viral expression vectors
can be concurrently introduced into a somatic cell; this represents
a preferred embodiment of the present invention.
[0081] In the method of the present invention, as genes that encode
reprogramming factors, for example, a combination of four kinds of
genes consisting of an Oct family gene, a Klf family gene, a Sox
family gene, and a Myc family gene can be used. A combination of
three kinds of genes consisting of an Oct family gene, a Klf family
gene, and a Sox family gene, or a combination of two kinds of genes
selected from among the aforementioned three kinds of genes can
also be used.
[0082] In the method of the present invention, it is preferable
that the above-described four kinds, three kinds, or two kinds of
genes be concurrently introduced into a somatic cell. To introduce
the above-described four kinds, three kinds, or two kinds of genes,
one kind of non-viral expression vector incorporating all these
genes may be used. Altenatively, several kinds of non-viral
expression vectors may be used in combination as appropriate, so as
to cover all the combinations of these genes. When several kinds of
non-viral expression vectors are used, it is preferable that
preferably two or three kinds, more preferably two kinds of
non-viral expression vectors be used. It is preferable that these
non-viral expression vectors be concurrently introduced into a
somatic cell.
[0083] If the number of genes introduced exceeds four kinds,
several kinds of non-viral expression vectors may be combined as
appropriate, so as to cover all the combinations of these genes.
When several kinds of non-viral expression vectors are used, it is
preferable that preferably two to five kinds, more preferably two
to four kinds, more preferably three or four of non-viral
expression vectors be used. These non-viral expression vectors are
preferably concurrently introduced into a somatic cell.
[0084] An example of a preferable method is a method wherein one
non-viral expression vector harboring an Oct family gene, a Klf
family gene, and a Sox family gene, and one non-viral expression
vector harboring a Myc family gene are introduced into a somatic
cell concurrently or at different times; in this method, it is
preferable that the two kinds of non-viral expression vectors be
concurrently introduced into the somatic cell. In another preferred
embodiment, it is also possible to use a method wherein one
non-viral expression vector harboring an Oct family gene, a Klf
family gene, a Sox family gene, and a Myc family gene is introduced
into a somatic cell.
[0085] In a preferred embodiment of the present invention, in a
combination of four kinds of genes consisting of an Oct3/4, Klf4,
Sox2, and c-Myc, or an optionally chosen combination of three kinds
or two kinds selected from among these four kinds of genes,
preferably the combination or three kinds or two kinds of genes,
wherein said combination does not contain c-Myc, can be used. This
preferred embodiment is hereinafter described specifically, to
which the scope of the present invention is never limited. [0086]
(a1) A method wherein one kind of non-viral expression vector, more
preferably a plasmid vector, harboring Oct3/4, Klf4, Sox2 and
c-Myc, is introduced into a somatic cell. [0087] (b1) A method
wherein a first non-viral expression vector, more preferably a
plasmid vector, harboring two kinds of genes selected from among
Oct3/4, Klf4, Sox2 and c-Myc, and a second non-viral expression
vector, more preferably a plasmid vector, harboring the remaining
two kinds of genes selected from among Oct3/4, Klf4, Sox2 and
c-Myc, are introduced into a somatic cell. Preferably, the first
non-viral expression vector and the second non-viral expression
vector can be concurrently introduced into a somatic cell. [0088]
(c1) A method wherein a first non-viral expression vector, more
preferably a plasmid vector, harboring three kinds of genes
selected from among Oct3/4, Klf4, Sox2 and c-Myc, and a second
non-viral expression vector, more a preferably a plasmid vector,
harboring the remaining one kind of gene selected from among
Oct3/4, Klf4, Sox2 and c-Myc, are introduced into a somatic cell.
Preferably, the first non-viral expression vector and the second
non-viral expression vector can be concurrently introduced into a
somatic cell. [0089] (d1) A method wherein a first non-viral
expression vector, more preferably a plasmid vector, harboring two
kinds of genes selected from among Oct3/4, Klf4 and Sox2, and a
second non-viral expression vector, more preferably a plasmid
vector, harboring the remaining one kind of gene selected from
among Oct3/4, Klf4 and Sox2, and c-Myc, are introduced into a
somatic cell. Preferably, the first non-viral expression vector and
the second non-viral expression vector can be concurrently
introduced into a somatic cell. [0090] (e1) A method wherein a
first non-viral expression vector, more preferably a plasmid
vector, harboring Oct3/4, Klf4 and Sox2, and a second non-viral
expression vector, more preferably a plasmid vector, harboring
c-Myc, are introduced into a somatic cell. Preferably, the first
non-viral expression vector and the second non-viral expression
vector can be concurrently introduced into a somatic cell. [0091]
(f1) A method wherein a first non-viral expression vector, more
preferably a plasmid vector, harboring two kinds of genes selected
from among Oct3/4, Klf4 and Sox2 in this order in the orientation
from the 5' to 3' end, and a second non-viral expression vector,
more preferably a plasmid vector, harboring c-Myc and any one gene
out of Oct3/4, Klf4 and Sox2 not contained in the first non-viral
expression vector, are introduced into a somatic cell. More
specifically, a first non-viral expression vector, preferably a
plasmid vector, harboring (i) Oct3/4 and Klf4, (ii) Klf4 and Sox2,
or (iii) Oct3/4 and Sox2 in this order in the orientation from the
5' to 3' end can be used; the first non-viral expression vector and
the second non-viral expression vector can be concurrently
introduced into a somatic cell. [0092] (g1) A method wherein a
first non-viral expression vector, more preferably a plasmid
vector, harboring Oct3/4, Klf4 and Sox2 in this order in the
orientation from the 5' to 3' end, and a second non-viral
expression vector, more preferably a plasmid vector, harboring
c-Myc are introduced into a somatic cell. Preferably, the first
non-viral expression vector and the. second non-viral expression
vector can be concurrently introduced into a somatic cell.
[0093] The method of (f1) or (g1) can be preferably used when the
somatic cell is derived from mouse.
[0094] In (b1) to (f2) above, for either one of the first non-viral
expression vector and the second non-viral expression vector, a
viral vector (e.g., retroviral vector, lentiviral vector,
adenoviral vector, adeno-associated viral vector, Sendai viral
vector or the like) can be used in place of the non-viral
expression vector.
[0095] In another preferred embodiment of the present invention, in
(a1) to (f2) above, L-Myc can be used in place of c-Myc.
[0096] In still another preferred embodiment, a combination of
three kinds of genes consisting of Oct3/4, Klf4 and Sox2 can be
used. This preferred embodiment is hereinafter described
specifically, to which the scope of the present invention is never
limited. [0097] (a2) A method wherein one kind of non-viral
expression vector, more preferably a plasmid vector, harboring
Oct3/4, Klf4 and Sox2, is introduced into a somatic cell. [0098]
(b2) A method wherein one kind of non-viral expression vector, more
preferably a plasmid vector, harboring Oct3/4, Klf4 and Sox2 in
this order in the orientation from the 5' to 3' end are introduced
into a somatic cell. [0099] (c2) A method wherein a first non-viral
expression vector, more preferably a plasmid vector, harboring two
kinds of genes selected from among Oct3/4, Klf4 and Sox2, and a
second non-viral expression vector, more preferably a plasmid
vector, harboring the remaining one kind of gene selected from
among Oct3/4, Klf4 and Sox2, are introduced into a somatic cell.
Preferably, the first non-viral expression vector and the second
non-viral expression vector can be concurrently introduced into a
somatic cell. [0100] (d2) A method wherein a first non-viral
expression vector; more preferably a plasmid vector, harboring two
kinds of genes selected from among Oct3/4, Klf4 and Sox2 in this
order in the orientation from the 5' to 3' end, and a second
non-viral expression vector, more preferably a plasmid vector,
harboring any one gene out of Oct3/4, Klf4 and Sox2 not contained
in the first non-viral expression vector are introduced into a
somatic cell. More specifically, a first non-viral expression
vector, preferably a plasmid vector, harboring (i) Oct3/4 and Klf4,
(ii) Klf4 and Sox2, or (iii) Oct3/4 and Sox2 in this order in the
orientation from the 5' to 3' end can be used, and the first
non-viral expression vector and the second non-viral expression
vector can be concurrently introduced into a somatic cell.
[0101] The method of (b2) or (d2) can be preferably used when the
somatic cell is derived from mouse.
[0102] In (c2) or (d2) above, for either one of the first non-viral
expression vector and the second non-viral expression vector, a
viral vector (e.g., retroviral vector, lentiviral vector,
adenoviral vector, adeno-associated viral vector, Sendai viral
vector or the like) can also be used in place of the non-viral
vector.
[0103] In still another preferred embodiment of the present
invention, a combination of two kinds of genes selected from among
Oct3/4, Klf4 and Sox2 can be used. This preferred embodiment is
hereinafter described specifically, to which the scope of the
present invention is never limited. [0104] (a3) A method wherein
one kind of non-viral expression vector, more preferably a plasmid
vector, harboring two kinds of genes selected from among Oct3/4,
Klf4 and Sox2, is introduced into a somatic cell. [0105] (b3) A
method wherein one kind of non-viral expression vector, more
preferably a plasmid vector, harboring (i) Oct3/4 and Klf4, (ii)
Klf4 and Sox2, or (iii) Oct3/4 and Sox2 in this order in the
orientation from the 5' to 3' end, is introduced into a . somatic
cell. [0106] (c3) A method wherein a first non-viral expression
vector, more preferably a plasmid vector, harboring one kind of
gene selected from among Oct3/4, Klf4 and Sox2, and a second
non-viral expression vector, more preferably a plasmid vector,
harboring any one gene out of Oct3/4, Klf4 and Sox2 not contained
in the first non-viral expression vector, are introduced into a
somatic cell. Preferably, the first non-viral expression vector and
the second non-viral expression vector can be concurrently
introduced into a somatic cell.
[0107] The method of (b3) can be preferably used when the somatic
cell is derived from mouse.
[0108] In (c3) above, for either one of the first non-viral
expression vector and the second non-viral expression vector, a
viral vector (e.g., retroviral vector, lentiviral vector,
adenoviral vector, adeno-associated viral vector, Sendai viral
vector or the like) can be used in place of the non-viral
vector.
[0109] In still another preferred embodiment of the present
invention, a combination of six kinds of genes selected from among
Oct3/4, Klf4, Sox2, c-Myc, Lin28 and Nanog can be used. This
preferred embodiment is hereinafter described specifically, to
which the scope of the present invention is never limited. (a4) A
method wherein a first non-viral expression vector, more preferably
a plasmid vector, harboring two kinds of genes selected from among
Oct3/4, Klf4 and Sox2, a second non-viral expression vector, more
preferably a plasmid vector, harboring the remaining one kind of
gene selected from among Oct3/4, Klf4 and Sox2, and a third
non-viral expression vector, more preferably a plasmid vector,
harboring c-Myc, Lin28 and Nanog genes are introduced into a
somatic cell. Preferably, the first, second and third non-viral
expression vectors can be concurrently introduced into a somatic
cell. [0110] (b4) A method wherein a first non-viral expression
vector, more preferably a plasmid vector, harboring (i) Oct3/4 and
Klf4, (ii) Klf4 and Sox2, (iii) Oct3/4 and Sox2 or (iv) Sox2 and
Klf4 in this order in the orientation from the 5' to 3' end, a
second non-viral expression vector, more preferably a plasmid
vector, harboring the remaining one kind of gene selected from
among Oct3/4, Klf4 and Sox2, and a third non-viral expression
vector, more preferably a plasmid vector, harboring c-Myc, Lin28
and Nanog genes in this order in the orientation from the 5' to 3'
end are introduced into a somatic cell.
[0111] When a gene encoding a factor that induces cell
immortalization, such as TERT, SV40 large T antigen, HPV16 E6,
HPV16 E7 or Bmil, is further combined with the two, three, four or
six genes mentioned above, it can be preferably incorporated into
another non-viral expression vector.
[0112] In the context above, when a plurality of genes (e.g., Oct
family gene, Klf family gene, and Sox family gene) are incorporated
in one kind of non-viral expression vector, these genes can
preferably be inserted into the non-viral expression vector with an
intervening sequence enabling polycistronic expression. By using an
intervening sequence enabling polycistronic expression, it is
possible to more efficiently express a plurality of genes
incorporated in one kind of non-viral expression vector. Useful
sequences enabling polycistronic expression include, for example,
the 2A sequence of foot-and-mouth disease virus (SEQ ID NO:61,
sometimes referred to as FMDV 2A-self-processing sequence) (PLoS
ONE 3, e2532, 2008; Stem Cells 25, 1707, 2007), IRES sequence and
the like, preferably the 2A sequence. More specifically, when a
non-viral expression vector harboring (i) Oct3/4, Klf4 and Sox2,
(ii) Oct3/4 and Klf4, (iii) Klf4 and Sox2, (iv) Oct3/4 and Sox2,
(v) Sox2 and Klf4 or (vi) c-Myc, Lin28 and Nanog in this order in
the orientation from the 5' to 3' end is constructed, it is
preferable to insert the 2A sequence between these genes.
Accordingly, the present invention also provides a use of the 2A
sequence for preparing a non-viral expression vector for iPS cell
induction, harboring two or more kinds of reprogramming
factors.
[0113] The number of repeats of the manipulation to introduce a
non-viral expression vector into a somatic cell is not particularly
limited, as far as the effect of the present invention of
reprogramming a somatic cell to produce an induced pluripotent stem
cell can be accomplished, the transfection can be performed once or
more optionally chosen times (e.g., once to 10 times, once to 5
times or the like). When two or more kinds of non-viral expression
vectors are introduced into a somatic cell, it is preferable that
these all kinds of non-viral expression vectors be concurrently
introduced into a somatic cell; however, even in this case, the
transfection can be performed once or more optionally chosen times
(e.g., once to 10 times, once to 5 times or the like), preferably
the transfection can be repeatedly performed twice or more (e.g., 3
times or 4 times).
[0114] When the transfection is repeated twice or more, the time
interval is exemplified by, but not limited to, 12 hours to 1 week,
preferably 12 hours to 4 days, for example, 1 day to 3 days.
[0115] As used herein, the term "induced pluripotent stem cell (iPS
cell)" refers to a cell possessing properties similar to that of ES
cells, more specifically including undifferentiated cells
reprogrammed from somatic cells possessing pluripotency and
proliferating (self-renewal) capability. It should be noted,
however, that this term must not be construed as limiting in any
sense, and must be construed in the broadest sense. A method of
preparing an induced pluripotent stem cell by means of hypothetical
nuclear reprogramming factors is described in WO2005/80598 (in this
publication, the term ES-like cell is used), and a method of
isolating an induced pluripotent stem cell is also described
specifically. WO2007/69666 discloses specific examples of
reprogramming factors and methods of somatic cell reprogramming
using the same. Therefore, it is desirable that in embodying the
present invention, those skilled in the art refer to these
publications.
[0116] In addition to the gene that encodes a reprogramming factor,
a regulatory sequence required for transcription (e.g., promoter,
enhancer, and/or terminator and the like) is preferably operably
linked to the gene in the non-viral expression vector.
[0117] As the promoter, a DNA sequence exhibiting transcription
activity in somatic cells can be used, and the promoter can be
chosen as appropriate according to animal species and kind of
somatic cell. Examples of useful promoters that can be expressed in
mammalian cells include a promoter of the IE (immediate early) gene
of cytomegalovirus (human CMV), initial promoter of SV40, promoter
of retrovirus, metallothionein promoter, heat shock promoter, SRa
promoter and the like. An enhancer of the IE gene of human CMV may
be used along with a promoter. A useful promoter is the CAG
promoter (comprising cytomegalovirus enhancer, chicken .beta.-actin
promoter and .beta.-globin gene polyA signal site).
[0118] The non-viral expression vector may incorporate a DNA
sequence that allows the autonomous replication of the expression
vector in a mammalian somatic cell. An example of the DNA sequence
is the SV40 replication origin.
[0119] The non-viral expression vector is preferably an expression
vector autonomously replicable outside the chromosome, and the
non-viral expression vector is preferably one that is not
integrated in the chromosome. More preferable examples include
plasmid vectors. Examples of the plasmid vector include, but are
not limited to, Escherichia coli-derived plasmids (ColE-series
plasmids such as pBR322, pUC18, pUC19, pUC118, pUC119, and
pBluescript, and the like), Actinomyces-derived plasmids (pIJ486
and the like), Bacillus subtilis-derived plasmids (e.g., pUB110,
pSH19 and others), yeast-derived plasmids (YEp13, YEp 24, Ycp50 and
the like) and the like, as well as artificial plasmid vectors and
the like.
[0120] Examples of easily available non-viral expression vectors
include, but are not limited to, pCMV6-XL3 (OriGene Technologies
Inc.), EGFP-C1 (Clontech), pGBT-9 (Clontech), pcDNAI (FUNAKOSHI),
pcDM8 (FUNAKOSHI), pAGE107 (Cytotechnology, 3,133, 1990), pCDM8
(Nature, 329, 840, 1987), pcDNAI/AmP (Invitrogen), pREP4
(Invitrogen), pAGE103 (J. Blochem., 101, 1307, 1987), pAGE210 and
the like.
[0121] The non-viral expression vector may incorporate a selectable
marker as required. Examples of the selectable marker include genes
that are deficient in the host cell, such as the dihydrofolate
reductase (DHFR) gene or the Schizosaccaromyces pombe TPI gene, and
genes for resistance to drugs such as ampicillin, kanamycin,
tetracycline, chloramphenicol, neomycin, or hygromycin.
[0122] While a non-viral expression vector such as plasmid vector
introduced into a somatic cell is typically not integrated into the
genome of the cell, under selection pressure for iPS cell
induction, increased integration efficiency of non-viral expression
vector may be observed due to the necessity of stable expression of
reprogramming factors. Accordingly, when the iPS cells of interest
are intended to use for regenerative medicine and the like, the
non-viral expression vector can preferably contain a sequence
enabling the excicion of transgenes, such as loxP sequence (Chang
et al., STEM CELLS Published Online: 12 Feb. 2009 (doi:
10.1002/stem.39)), piggyback transposon (Kaji et al., Nature
advance online publication 1 Mar. 2009 (doi:10.1038/nature07864);
Woltjen et al., Nature advance online publication 1 Mar. 2009
(doi:10.1038/nature07863)) and tetracycline responsive element in
promoter region (Tet-OnR & Tet-Off R Gene Expression Systems,
Clontech).
[0123] A method of ligating a gene that encodes a reprogramming
factor, a promoter, an enhancer, and/or a terminator and the like,
used in the present invention, in an appropriate order to construct
a non-viral expression vector capable of expressing the
reprogramming factor in the somatic cell, is obvious to those
skilled in the art.
[0124] When two or more kinds of genes that encode reprogramming
factors are used, the genes may be incorporated in one non-viral
expression vector. Alternatively, two or more kinds of non-viral
expression vectors incorporating different genes may be used. In
the latter case, one non-viral expression vector incorporating two
or more kinds of genes and a non-viral expression vector
incorporating one or more kind genes different therefrom can be
combined as appropriate.
[0125] Any method of expression vector introduction into an animal
cell available to those skilled in the art can be used to introduce
a non-viral expression vector into a somatic cell. Examples of
useful methods include the use of a transfection reagent such as
the FuGENE 6 transfection reagent (Roche), the use of a
microporator, the electroporation method, the calcium phosphate
method, the lipofection method, the DEAE-dextran-mediated
transfection method, the transfection method, the microinjection
method, the cationic lipid-mediated transfection method, and the
like. Nucleofection can also be used to introduce a gene. These
methods may be used in combination.
[0126] In introducing a non-viral expression vector into a somatic
cell, the expression vector may be introduced into the somatic cell
being cultured on feeder cells, and may be introduced only into the
somatic cell. To increase expression vector introduction
efficiency, the latter method is sometimes suitable. The feeder
cells used may be those for cultivation of embryonic stem cells;
for example, primary culture fibroblasts from a 14- to 15-day mouse
embryo, STO (fibroblast-derived cell line) and the like, treated
with a chemical agent such as mitomycin C or exposed to radiation,
and the like can be used.
[0127] By culturing a somatic cell incorporating a non-viral
expression vector under appropriate conditions, it is possible to
allow nuclear reprogramming to progress autonomically, and to
produce an induced pluripotent stem cell from the somatic cell. The
step of culturing a somatic cell incorporating a non-viral
expression vector to obtain an induced pluripotent stem cell can be
performed in the same manner as a conventional method using a
retrovirus; for example, this can be achieved as described in
publications such as Cell, 126, pp. 1-14, 2006; Cell, 131, pp.
1-12, 2007; and Science, 318, pp. 1917-1920, 2007. In producing a
human induced pluripotent stem cell, it is sometimes desirable that
the cell culture density after expression vector introduction be
set at a level lower than that for ordinary animal cell culture.
For example, it is preferable to continue the cultivation at a cell
density of 10,000 to 100,000 cells, preferably about 50,000 cells
per cell culture dish. Any medium can be used for the cultivation,
chosen as appropriate by those skilled in the art; for example, in
producing a human induced pluripotent stem cell, it is sometimes
preferable to use a medium suitable of human ES cell culture.
Regarding the choice of medium and culturing conditions, the
aforementioned publications serve for references.
[0128] The resulting induced pluripotent stem cells can be
identified using various markers characteristic of undifferentiated
cells; means for this identification are also described in the
aforementioned publications specifically and in detail. Various
media allowing the maintenance of undifferentiated state and
pluripotency of ES cells or media not allowing the maintenance of
these properties are known in the art; by using appropriate media
in combination, an induced pluripotent stem cell can be isolated
efficiently. The differentiation potential and proliferation
potential of the isolated induced pluripotent stem cells are easily
confirmable for those skilled in the art by utilizing a method of
identification in common use for ES cells. When the resulting
induced pluripotent stem cell is proliferated under appropriate
conditions, a colony of induced pluripotent stem cells is obtained;
it is possible to identify the presence of an induced pluripotent
stem cell on the basis of the shape of the colony. For example, it
is known that mouse induced pluripotent stem cells form raised
colonies, whereas human induced pluripotent stem cells form flat
colonies, and the shapes of these colonies are extremely similar to
those of mouse ES cell and human ES cell colonies, respectively;
therefore, it is possible for those skilled in the art to identify
the resulting induced pluripotent stem cell on the basis of the
shape of the colony. When reprogramming is performed using a
somatic cell having a gene incorporating a marker gene such as GFP
downstream of a promoter of gene specifically expressing in ES
cells, it is possible to identify an induced pluripotent stem cell
if the cell becomes positive for the marker (GFP).
[0129] "Somatic cells" to be reprogrammed by the method of the
present invention refers to any cells except totipotent and
pluripotent cells such as early embryos and ES cells, and the
choice thereof is not limited. For example, as well as somatic
cells in the fetal stage, neonatal somatic cells and mature somatic
cells may be used. Preferably, somatic cells derived from mammals,
including humans, are used; more preferably human- or mouse-derived
somatic cells are used. Specifically, (1) tissue stem cells
(somatic stem cells) such as nerve stem cells, hematopoietic stem
cells, mesenchymal stem cells, and dental pulp stem cells, (2)
tissue progenitor cells, or (3) io differentiated cells such as
lymphocytes, epithelial cells, muscle cells, fibroblasts (dermal
cells and the like), hair cells, liver cells, and gastromucosal
cells can be mentioned. When an induced pluripotent stem cell is
used to treat a disease, it is desirable to use somatic cells
separated from a patient to be treated or from another person
sharing the same type of HLA as that of the patient; for example,
somatic cells involved in disease and somatic cells involved in
disease treatment and the like can be used.
[0130] In the present invention, to increase the efficiency of
induced pluripotent stem cell establishment, in addition to the
introduction of a non-viral expression vector of the present
invention, various establishment efficiency improvers may be
introduced or added. Examples of iPS cell establishment efficiency
improvers include, but are not limited to, histone deacetylase
(HDAC) inhibitors [e.g., valproic acid (VPA) (Nat. Biotechnol.,
26(7): 795-797 (2008)), low-molecular inhibitors such as
trichostatin A, sodium butyrate, MC 1293, and M344, nucleic
acid-based expression inhibitors such as siRNA and shRNA against
HDAC (e.g., HDAC1 siRNA Smartpool.RTM. (Millipore), HuSH 29 mer
shRNA Constructs against HDAC1 (OriGene) and the like), and the
like], G9a histone methyltransferase inhibitors [e.g.,
low-molecular inhibitors such as BIX-01294 (Cell Stem Cell, 2:
525-528 (2008)), nucleic acid-based expression inhibitors such as
siRNA and shRNA against G9a (e.g., G9a siRNA (human) (Santa Cruz
Biotechnology) and the like) and the like], L-channel calcium
agonist (e.g., Bayk8644) (Cell Stem Cell, 3, 568-574 (2008)), UTF1
(Cell Stem Cell, 3, 475-479 (2008)), Wnt Signaling (e.g., soluble
Wnt3a) (Cell Stem Cell, 3, 132-135 (2008)), 2i/LIF (2i is an
inhibitor of mitogen-activated protein kinase signaling and
glycogen synthase kinase-3; PloS Biology, 6(10), 2237-2247 (2008)),
p53 inhibitors (e.g., siRNA and shRNA against p53 (Cell Stem Cell,
3, 475-479 (2008)) and the. like. The nucleic acid-based expression
inhibitors may be in the form of expression vectors harboring a DNA
that encodes siRNA or shRNA. In this case, the DNA that encodes
siRNA or shRNA may be inserted into a non-viral expression vector
of the present invention, together with reprogramming factors.
[0131] The induced pluripotent stem cell produced by the method of
the present invention is not subject to limitations concerning the
use thereof, and can be used for all types of studies and
investigations with the use of ES cells and for the treatment of
diseases using ES cells, in place of ES cells. For example, by
treating an induced pluripotent stem cell obtained from a somatic
cell collected from a patient by the method of the present
invention with retinoic acid, a growth factor such as EGF, or
glucocorticoid and the like, desired differentiated cells (e.g.,
nerve cells, myocardial cells, blood cells and the like) can be
induced to form an appropriate tissue. By returning the
differentiated cell or tissue thus obtained to the patient, stem
cell therapy by autologous cell transplantation can be
accomplished. It should be noted that the use of an induced
pluripotent stem cell of the present invention is not limited to
the above-described particular embodiment.
[0132] The present invention also provides a non-viral expression
vector for use in the above-described method of producing an
induced pluripotent stem cell, i.e., a non-viral expression vector
(preferably a plasmid vector) incorporating at least one gene that
encodes a reprogramming factor. The structure of the vector is as
described in detail in the section of a method of producing an
induced pluripotent stem cell of the present invention.
[0133] An example is a non-viral expression vector incorporating an
Oct family gene, a Kif family gene, and a Sox family gene,
preferably incorporated in this order in the orientation from the
5' to 3' end. A more preferable example is a non-viral expression
vector incorporating these genes with an intervening sequence
enabling polycistronic expression, particularly preferably a
non-viral expression vector wherein OCT3/4, Klf4 and Sox 2 are
incorporated with an intervening sequence enabling polycistronic
expression, preferably FMDV 2A-self-processing sequence, in this
order in the orientation from the 5' to 3' end.
[0134] Since a non-viral expression vector such as plasmid vector
introduced into a somatic cell is typically not integrated into the
genome of the cell, in a preferred embodiment, the present
invention provides an induced pluripotent stem cell wherein
transgenes are not integrated into the genome. Since such iPS cell
reduces a risk causing tumorigenesis in tissues or organs
differentiated therefrom. and/or disturbance (e.g., disruption or
activation) of an endogenous gene, it can preferably be used for
regenerative medicine such as cell transplantation therapy.
[0135] However, under selection pressure for iPS cell induction,
increased integration efficiency of non-viral expression vector can
be observed due to the necessity of stable expression of
reprogramming factors. Therefore, in another preferred embodiment,
the present invention provides an induced pluripotent stem cell
wherein transgenes are integrated into the genome in the form of
plasmid. Such iPS cell can reduce a risk causing tumorigenesis in
tissues or organs differentiated therefrom as compared to an iPS
cell induced by retroviral infection. In addition, the transgenes
can be excised from the genome as necessary using a Cre/loxP system
(Chang et al., 2009 (supra)) or a piggyback transposon vector and
piggyback transposon (Kaji et al., 2009 (supra); Woltjen et al.,
2009 (supra)) or tetracycline dependent gene induction. A Cre
recombinase or transposase for the excision can be introduced into
and expressed in the iPS cell using a plasmid vector or adenoviral
vector. In the case of using tetracycline dependent gene induction,
Tet-repressor protein or mutated Tet-repressor protein is
concomitantly expressed.
[0136] The present invention is hereinafter described in more
detail by means of the following Examples, which, however, are not
to be construed as limiting the scope of the invention.
Example 1
[0137] Mice having a Nanog reporter were used as an experimental
system (Okita et al. Nature, Vol. 448, pp. 313-317, 2007). These
mice were prepared by incorporating EGFP and a puromycin resistance
gene into the Nanog gene locus of a BAC (bacterial artificial
chromosome) purchased from BACPAC Resources. The mouse Nanog gene
is expressed specifically in pluripotent cells such as ES cells and
early embryos. Mouse iPS cells positive for this reporter have been
shown to possess a differentiation potential nearly equivalent to
that of ES cells. These Nanog reporter mice were mated with Fbx15
reporter mice (Tokuzawa et al. Mol Cell Biol, Vol. 23, 2699-2708
(2003)), whereby mutant mice having both the Nanog reporter and the
Fbx15 reporter were generated.
[0138] The plasmid used for reprogramming was prepared by treating
pCX-EGFP (a plasmid supplied by Dr. Masaru Okabe at Osaka
University: FEBS Letters, 407, 313-319, 1997) with EcoRI, and
inserting a construct wherein the coding regions of Oct3/4, Sox2,
and Klf4 (all mouse-derived genes) are ligated via the 2A sequence
of foot-and-mouth disease virus in the order of Oct3/4, Klf4, and
Sox2, in place of EGFP (pCX-2A-mOKS; FIG. 2). Likewise, a plasmid
with the coding region of c-Myc inserted thereinto was prepared
(pCX-c-Myc; FIG. 2).
[0139] In preparing the construct of the 2A sequence and Oct3/4,
Klf4, and Sox2 ligated together, first, sense and antisense
oligonucleotides comprising the 2A sequence of foot-and-mouth
disease virus (SEQ ID NO:61), upstream restriction endonuclease
sites (XbaI and BglII), and downstream restriction endonuclease
sites (BspHI, Mfel and PstI), were annealed and inserted into
pBluescript II KS (-) vector digested with the XbaI and PstI
(pBS-2A). Subsequently, a mouse cDNA that encodes Oct3/4 or Klf4
was amplified by PCR, the translation termination codon was
replaced with a BamHI site, and each cDNA was cloned into pCR2.1.
Subsequently, the cDNAs of Oct3/4 and Klf4 were ligated with pBS-2A
using an appropriate restriction endonuclease to yield
pBS-Oct3/4-2A and pBS-Klf4-2A. Subsequently, Klf4-2A was inserted
into pBS-Oct3/4-2A in frame using an appropriate restriction
endonuclease, whereby pBS-Oct3/4-2A-Klf4-2A was produced.
Subsequently, the resulting Oct3/4-2A-Klf4-2A construct was ligated
with a cDNA of Sox2 having a translation termination codon in
frame, using an appropriate restriction endonuclease. Finally, the
resulting Oct3/4-2A-Klf4-2A-Sox2-STOP construct, wherein the 2A
sequences and Oct3/4, Klf4, and Sox2 were ligated together, was
inserted into the EcoRI site of pCX-EGFP, whereby pCX-2A-mOKS was
prepared.
[0140] Fibroblasts (MEF) were isolated from the aforementioned
mutant mouse fetus (13.5 days after fertilization). Not expressing
the Nanog gene, MEF does not express EGFP producing green
fluorescence. As such, the MEFs were sown to a 6-well culture plate
(Falcon), previously coated with 0.1% gelatin (Sigma), at
1.3.times.10.sup.5 cells per well. The culture medium used being
DMEM/10% FCS (DMEM (Nacalai Tesque) supplemented with 10% fetal
calf serum), the MEFs were cultured at 37.degree. C., 5% CO.sub.2.
The following day, 4.5 .mu.L of the FuGene6 transfection reagent
(Roche) was added in 100 .mu.L of Opti-MEM I Reduced-Serum Medium
(Invitrogen), and the medium was allowed to stand at room
temperature for 5 minutes. Thereafter, 1.5 .mu.g of an expression
vector (pCX-2A-mOKS) was added, and the medium was allowed to stand
at room temperature for 15 minutes, after which the medium was
added to a MEF culture medium. The following day, the medium was
removed, and 1.5 .mu.g of another expression vector (pCX-c-Myc) was
introduced with the FuGene6 transfection reagent as described
above.
[0141] The following day, the culture medium was replaced with a
fresh supply (DMEM/10% FCS) and an expression vector (pCX-2A-mOKS)
was introduced as described above; the day after, the culture
medium was replaced with an ES cell culture medium (DMEM (Nacalai
Tesque) supplemented with 15% fetal calf serum, 2 mM L-glutamine
(Invitrogen), 100 .mu.M non-essential amino acids (Invitrogen), 100
.mu.M 2-mercaptoethanol (Invitrogen), 50 U/mL penicillin
(Invitrogen) and 50 mg/mL streptomycin (Invitrogen)), and an
expression vector (pCX-c-Myc) was introduced using the FuGene6
transfection reagent as described above.
[0142] The following day, the medium was replaced with an ES cell
culture medium. On day 9 after sowing, the MEF culture medium was
removed, and the cells were washed by the addition of PBS 2 mL.
After the PBS was removed, 0.25% Trypsin/1 mM EDTA (Invitrogen) was
added, and the reaction was carried out at 37.degree. C. for about
5 minutes. After cells rose, an ES cell culture medium was added,
the cells were suspended, and 1.times.10.sup.6(Exp432A) or
2.times.10.sup.5 (Exp432B) cells were sown onto a 100 mm dish with
feeder cells sown thereto previously. The feeder cells used were
SNL cells that had been treated with mitomycin C to terminate their
cell division.
[0143] Subsequently, the ES cell culture medium was replaced with a
fresh supply every two days until a visible colony emerged;
colonization began around day 17, and complete colonization was
observed around day 24 (FIG. 1). The time schedule above is
summarized in Exp432 in FIGS. 1 and 3.
[0144] The cells obtained became GFP-positive gradually, exhibited
a morphology indistinguishable from that of mouse ES cells (432A-1
in FIG. 4), tested positive for various ES cell markers at similar
levels as with ES cells (iPS-432A-1 in FIG. 5), and produced adult
chimeric mice. Based on the colony shape characteristic of mouse
iPS cells and GFP-positive results and results positive for other
non-differentiation markers, it was concluded that by introducing
the above-described expression vector into MEF cells, nuclear
reprogramming was completely advanced to produce an iPS cell, and
the iPS cell proliferated and formed the visible colony. Hence,
these results showed that an iPS cell could be prepared without
using a retrovirus or a lentivirus. PCR analysis detected the
integration of the above-described expression vector into the host
genome (iPS-432A-1 in FIG. 6).
Example 2
[0145] To avoid the integration of pCX-2A-mOKS and pCX-c-Myc into
the host genome, the transfection protocol was modified. On days 1,
3, 5, and 7 after the start of the experiment, pCX-2A-mOKS and
pCX-c-Myc were transfected together (Exp440 in FIG. 3). As a
result, many GFP-positive colonies were obtained, and cells
morphologically indistinguishable from ES cells were produced
(440A-3 in FIG. 4). The cells obtained expressed the ES cell
markers at the same level as with ES cells (iPS-440A in FIG. 5). To
examine for the integration of the plasmid DNA into the genome, 16
sets of PCR primers capable of amplifying each portion of the
plasmid were designed (FIGS. 2, 13 and 14). In 9 of the 11
GFP-positive clones obtained by the modified protocol, no
amplification of an exogenous DNA was observed (FIG. 6).
Furthermore, in Southern blot analysis, no integration of an
exogenous gene was detected in these clones (FIG. 11). Although the
possible presence of a small plasmid fragment cannot be ruled out
definitely, the above results showed that these iPS cells did not
have the pCX-2A-mOKS and pCX-c-Myc plasmids integrated into the
host genome.
[0146] To rule out the possibility that the iPS cells without
integration are derived from possibly contaminating Nanog-GFP ES
cells, SSLP analysis was performed. In Exp440 in FIG. 3, MEF cells
from five fetuses were used. In the SSLP analysis, these five
fetuses were distinguishable, and the derivations of the iPS cells
without integration were identified (FIG. 12). This analysis also
showed that the iPS cells without integration differed from the ES
cells derived from the 129S4 strain (FIG. 12).
Example 3
[0147] To confirm the pluripotency of iPS cells without
integration, iPS cells obtained as described in Example 2 were
subcutaneously transplanted to nude mice. All clones tested
(440A-3, -4, -8 and -10) produced tumors, which included a broad
range of cell types, including cells derived from all the three
germ layers (FIG. 7). Furthermore, iPS cells without integration
were injected into ICR mouse blastocysts. Judging from the coat
colors, adult chimeras were obtained from all clones injected
(440A-3, -4, -6, -8, -9 and -10) (FIG. 8). In these chimeric mice,
PCR analysis did not detect the integration of any of the
transgenes (FIG. 9). The PCR analysis detected both the Nanog and
Fbx15 reporters in the chimeras (FIG. 9). Combined with the fact
that iPS cells without integration emerged from the double reporter
mice, and that the inventor's laboratory does not keep double
reporter ES cells, these results showed that the chimeras were
derived from iPS cells without integration, rather than from
contaminating ES cells. Hence, these results confirmed that the iPS
cells without integration possessed pluripotency.
[0148] Long-term examination of 71 chimeric mice obtained and
offspring thereof showed that in the chimeric mice derived from an
iPS cell prepared by introducing 4 genes (Oct3/4, Klf4, Sox2,
c-Myc) using a retrovirus, and offspring thereof, compared with
normal mice, the mortality rate began to rise earlier, whereas the
chimeric mice derived from an iPS cell without integration of the 4
genes and offspring thereof exhibited a survival curve similar to
that of normal mice.
[0149] When chimeric mice obtained and wild mice were mated, F1
mice were obtained; therefore, it was confirmed that iPS cells
without integration contributed to the germline
(germline-transmission).
Example 4
[0150] Human dental pulp stem cells (clone name; DP31,
PCT/JP2008/068320, J. Dent. Res., 87(7):676-681 (2008)) were used
as an experimental system. The DP31 was allowed to express the
mouse ecotropic virus receptor Slc7a1 gene using a lentivirus as
described in Cell, 131, 861-872 (2007). These cells were cultured
using the MSCGM bullet kit (Lonza).
[0151] The plasmids used for reprogramming were prepared from
pCX-EGFP (supplied by Dr. Masaru Okabe at Osaka University, FEBS
Letters, 407, 313-319, 1997) in the same manner as Example 1.
Specifically, the pCX-EGFP was treated with EcoRI, and a construct
with the coding regions of SOX2 and KLF4 ligated via the 2A
sequence of foot-and-mouth disease virus therein was inserted in
place of EGFP, whereby the plasmid pCX-hSK was prepared. Likewise,
a plasmid with c-Myc, Lin28, and Nanog ligated via the 2A sequence
(pCX-hMLN) therein, a plasmid with the OCT3/4 coding region
inserted therein (pCX-hOCT3/4), and a plasmid with the SV40 Large T
antigen inserted therein (pCX-SV40LT) were prepared.
[0152] The DP31 cultured in a 100 mm dish was washed with PBS,
0.25% Trypsin/1 mM EDTA (Invitrogen) was added, and the reaction
was carried out at 37.degree. C. for about 5 minutes. After cells
rose, MSCGM was added, the cells were suspended, and
6.times.10.sup.5 cells were recovered in a 15 mL tube. The cells
were centrifuged at 800 rpm for 5 minutes; after the supernatant
was removed, and the expression plasmids were introduced using the
Human Dermal Fibroblast Nucleofector Kit (Amaxa). The amounts of
plasmids used were 0.5 .mu.g for pCX-hOCT3/4, 1.0 .mu.g for
pCX-hSK, 1.5 .mu.g for pCX-hMLN, and 0.5 .mu.g for pCX-SV40LT.
After the treatment, the cells were sown to a 6-well plate. After
being cultured with MSCGM for 10 days, the cells were again washed
with PBS, 0.25% Trypsin/1 mM EDTA (Invitrogen) was added, and the
reaction was carried out at 37.degree. C. for about 5 minutes.
After cells rose, MSCGM was added, the cells were suspended, and
1.times.10.sup.6 cells were sown onto a 100 mm dish with feeder
cells sown thereto previously. The feeder cells used were SNL cells
that had been treated with mitomycin C to terminate their cell
division. Thereafter, until a colony began to be observed, the
medium was replaced with a fresh supply every two days. The medium
used was prepared by mixing equal volumes of a primate ES cell
culture medium (ReproCELL) supplemented with MSCGM and bFGF (4
ng/mL), respectively. Colonization began around day 19, confirming
the establishment of human iPS cell (FIG. 15).
[0153] Next, fetal human HDF (Cell applications, INC) was
transfected with the same seven kinds of genes as described above.
After the transfection, the cells were cultured using a primate ES
cell culture medium (ReproCELL) supplemented with 4 ng/ml
recombinant human bFGF (WAKO). MSTO cells served as feeder cells.
Photographs of cells on day 31 after transfection (5 clones: 203A-1
to 203A-5, of which 203A-4 was picked up as a negative control) are
shown in FIG. 16, and photographs of cells in the 2nd subculture
are shown in FIG. 17. The 203A-1 to 203A-3 and 203A-5 clones
exhibited a typical ES cell-like morphology, confirming the
establishment of human iPS cells.
[0154] These cells were subjected to genomic-PCR analysis, and
examined for the integration of the transgenes into the genome. The
results are shown in FIG. 18. In all clones, the integration of
Oct3/4 (pCX-hOCT3/4) and c-Myc (pCX-hMLN) was detected. The
integration of Klf4 (pCX-hSK) was detected in the clones other than
203A-4. The integration of SV40LT (pCX-SV40LT) was not detected in
any of the clones.
Example 5
[0155] Dental pulp stem cells DP31, used in Example 4, were
transfected with six kinds of genes, excluding the SV40 Large T
antigen (pCX-hSK, pCX-hMLN, pCX-hOCT3/4), in the same manner as
Example 4. Photographs of cells on day 35 after the transfection (5
clones: 217A-1 to -4 and -6) are shown in FIG. 19. Photographs of
cells in the 2nd subculture are shown in FIG. 20. All clones
exhibited a typical ES cell-like morphology, confirming the
establishment of human iPS cells.
[0156] These human iPS cell clones established (217A-1 to 217A-4,
217A-6) were subjected to genomic-PCR analysis. The results are
shown in FIG. 21. In all these clones, the integration of the
transgenes was demonstrated.
Example 6
[0157] An HDF cell line derived from a 6-year-old Japanese female
(HDF-120; JCRB) was allowed to express the Slc7a1 gene. The
resulting cells (HDF-120-Slc) were transfected with the
aforementioned six kinds of genes and an shRNA against p53 (shRNA2:
SEQ ID NO:62) (vectors introduced: pCX-hOCT3/4, pCX-hSK,
pCX-hMLN-shp53).
[0158] Each of pCX-hOCT3/4 (0.5 .mu.g), pCX-hSK (1.0 .mu.g), and
pCX-hMLN-shp53 (1.5 .mu.g) was electrically introduced into
6.0.times.10.sup.5 cells of HDF-120-Slc using Microporator (100
.mu.L tip, 1600 V, 10 ms, 3 times). Ten days later, each vector was
once again electrically introduced under the same conditions, and
the cells were sown onto MSTO (100 mm dish). These cells were
cultured using DMEM/10% FCS until day 10, thereafter using a
primate ES cell culture medium (ReproCELL) supplemented with 4
ng/ml recombinant human bFGF (WAKO). Photographs of cells on day 35
after the first electroporation are shown in FIG. 22. Photographs
of cells after passage culture are shown in FIG. 23. A typical ES
cell-like morphology was exhibited, confirming the establishment of
human iPS cells. Genomic-PCR analysis demonstrated the integration
of the transgenes (lane 279A-2 in FIG. 24).
Example 7
[0159] Expression vectors separately incorporating the four kinds
of genes Oct3/4, Klf4, Sox2 and c-Myc (pCX-Oct4, pCX-Sox2,
pCX-Klf4, pCX-c-Myc) were introduced into MEF cells derived from a
Nanog reporter mouse (Okita et al. Nature, Vol. 448, pp. 313-317,
2007) per the protocol in Example 2.
[0160] First, the Nanog reporter MEF cells were sown onto a
gelatin-coated 6-well plate (1.3.times.10.sup.5 cells/well), and
transfected with each of pCX-Oct4 (0.37 .mu.g), pCX-Sox2 (0.36
.mu.g), pCX-Klf4 (0.39 .mu.g), and pCX-c-Myc (0.38 .mu.g) using
FuGene6 on days 1, 3, 5, and 7. On day 9, 1.times.10.sup.6 cells
(1.0) or 0.2.times.10.sup.6 cells (0.2) were sown onto MSTO-PH or
gelatin (100-mm dish), and colonies were selected on day 25.
Photographs of cells after the selection are shown in FIG. 25. A
colony shape characteristic of mouse iPS cells and GFP-positive
results were obtained, confirming the establishment of mouse iPS
cells. The mouse iPS cell clones established (497A-1 to A-5) were
subjected to genomic-PCR analysis. The results are shown in FIG.
26. Both 497A-2 and 497A-5 were shown to be iPS cells without
integration of any of the exogenous genes.
INDUSTRIAL APPLICABILITY
[0161] According to the method of the present invention, it is
possible to prepare a highly safe induced pluripotent stem cell
from, for example, a patient's somatic cell. The cells obtained by
differentiating the induced pluripotent stem cell (e.g., myocardial
cells, insulin-producing cells, nerve cells and the like) can be
safely used for stem cell transplantation therapy for a broad range
of diseases, including heart failure, insulin-dependent diabetes,
Parkinson's disease and spinal injury.
[0162] While the present invention has been described with emphasis
on preferred embodiments, it is obvious to those skilled in the art
that the preferred embodiments can be modified. The present
invention intends that the present invention can be embodied by
methods other than those described in detail in the present
specification. Accordingly, the present invention encompasses all
modifications encompassed in the gist and scope of the appended
"Claims".
[0163] The contents disclosed in any publication cited here,
including patents and patent applications, are hereby incorporated
in their entireties by reference, to the extent that they have been
disclosed herein.
[0164] This application is based on U.S. provisional patent
application Nos. 61/071,508, 61/136,246, 61/136,615 and 61/193,363,
the contents of which are hereby incorporated by reference.
Sequence CWU 1
1
62120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for G3PDH 1accacagtcc atgccatcac
20220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for G3PDH 2tccaccaccc tgttgctgta
20321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for Nanog 3agggtctgct actgagatgc t
21424DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for Nanog 4caacacctgg tttttctgcc accg
24524DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for Rex1 5acgagtggca gtttcttctt ggga
24624DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for Rex1 6tatgactcac ttccaggggg cact
24728DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for ECAT1 7tgtggggccc tgaaaggcga gctgagat
28828DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for ECAT1 8atgggccgcc atacgacgac gctcaact
28924DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for ERas 9actgcccctc atcagactgc tact
241024DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for ERas 10cactgccttg tactcgggta gctg
241120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for Fbx15 11gttggaatct gcttctacag
201220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for Fbx15 12cttcaccaag atttccgatg
201324DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for Esg1 13gaagtctggt tccttggcag gatg
241420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for Esg1 14actcgataca ctggcctagc
201524DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for Oct3/4 15ctgagggcca ggcaggagca cgag
241624DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for Oct3/4 16ctgtagggag ggcttcgggc actt
241724DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for Sox2 17ggttacctct tcctcccact ccag
241821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for Sox2 18tcacatgtgc gacaggggca g
211930DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for Klf4 19caccatggac ccgggcgtgg ctgccagaaa
302027DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for Klf4 20ttaggctgtt cttttccggg gccacga
272124DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for c-Myc 21cagaggagga acgagctgaa gcgc
242228DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for c-Myc 22ttatgcacca gagtttcgaa gctgttcg
282329DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for O-1 23cggaattcaa ggagctagaa cagtttgcc
292422DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for O-1 24ctgaaggttc tcattgttgt cg
222520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for O-2 25gatcactcac atcgccaatc
202622DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for O-2 26ctgggaaagg tgtcctgtag cc
222725DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for K 27gcgggaaggg agaagacact gcgtc
252824DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for K 28taggagggcc gggttgttac tgct
242923DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for K-S 29ccttacacat gaagaggcac ttt
233023DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for K-S 30cagctccgtc tccatcatgt tat
233125DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for M 31acactccccc aacaccagga cgttt
253229DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for M 32gctcgcccaa atcctgtacc tcgtccgat
293325DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for M 33gagatgagcc cgactccgac ctctt
253418DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for 1 34aggtgcaggc tgcctatc 183521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer for 1
35ttagccagaa gtcagatgct c 213620DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer for 2 36tggcgtaatc
atggtcatag 203723DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer for 2 37gcaacgcaat taatgtgagt tag
233821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for 3 38ctggatccgc tgcattaatg a
213917DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for 3 39ccgagcgcag cgagtca 174021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer for 4
40gccttatccg gtaactatcg t 214118DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer for 4 41gcaccgccta catacctc
184222DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for 5 42agttgcctga ctccccgtcg tg
224320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for 5 43ggagccggtg agcgtgggtc 204424DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer for 6
44ccgatcgttg tcagaagtaa gttg 244522DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer for 6
45tcacagaaaa gcatcttacg ga 224624DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer for 7 46gaaaagtgcc
acctggtcga catt 244724DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer for 7 47gggccattta ccgtaagtta
tgta 244819DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for 8 48tatcatatgc caagtacgc 194922DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer for 8
49tagatgtact gccaagtagg aa 225019DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer for 9 50tctgactgac
cgcgttact 195122DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer for 9 51agaaaagaaa cgagccgtca tt
225221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for 10 52gggggctgcg aggggaacaa a
215321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for 10 53gccgggccgt gctcagcaac t
215424DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for 11 54gcgagccgca gccattgcct ttta
245523DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for 11 55cccagatttc ggctccgcca gat
235630DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for Nanog-reporter 56tgggatccct atgctactcc
gtcgaagttc 305728DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer for Nanog-reporter 57ctaggcaaac
tgtggggacc aggaagac 285828DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer for Fbx15-reporter
58tggtccaaca tcttatacac agtaatga 285928DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer for
Fbx15-reporter 59gtggaactcc cttctagccc tctatccc 286026DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer for
Fbx15-reporter 60aatgggctga ccgcttcctc gtgctt 266181DNAArtificial
SequenceDescription of Artificial Sequence Synthetic 2A sequence
61aaaattgtcg ctcctgtcaa acaaactctt aactttgatt tactcaaact ggctggggat
60gtagaaagca atccaggtcc a 816248DNAArtificial SequenceDescription
of Artificial Sequence Synthetic shRNA against p53 62gactccagtg
gtaatctact gctcgagcag tagattacca ctggagtc 48
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