U.S. patent application number 12/656908 was filed with the patent office on 2010-08-19 for nuclear reprogramming factor and induced pluripotent stem cells.
This patent application is currently assigned to Kyoto University. Invention is credited to Shinya Yamanaka.
Application Number | 20100210014 12/656908 |
Document ID | / |
Family ID | 42983923 |
Filed Date | 2010-08-19 |
United States Patent
Application |
20100210014 |
Kind Code |
A1 |
Yamanaka; Shinya |
August 19, 2010 |
Nuclear reprogramming factor and induced pluripotent stem cells
Abstract
The present invention relates to a nuclear reprogramming factor
having an action of reprogramming a differentiated somatic cell to
derive an induced pluripotent stem (iPS) cell. The present
invention also relates to the aforementioned iPS cells, methods of
generating and maintaining iPS cells, and methods of using iPS
cells, including screening and testing methods as well as methods
of stem cell therapy. The present invention also relates to somatic
cells derived by inducing differentiation of the aforementioned iPS
cells.
Inventors: |
Yamanaka; Shinya; (Kyoto,
JP) |
Correspondence
Address: |
GREENBLUM & BERNSTEIN, P.L.C.
1950 ROLAND CLARKE PLACE
RESTON
VA
20191
US
|
Assignee: |
Kyoto University
Kyoto
JP
|
Family ID: |
42983923 |
Appl. No.: |
12/656908 |
Filed: |
February 18, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12213035 |
Jun 13, 2008 |
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12656908 |
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PCT/JP2006/324881 |
Dec 6, 2006 |
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12213035 |
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61001108 |
Oct 31, 2007 |
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60996289 |
Nov 9, 2007 |
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Current U.S.
Class: |
435/366 ;
435/377 |
Current CPC
Class: |
A61P 43/00 20180101;
C12N 5/0696 20130101; C07K 14/4702 20130101; C12N 2501/603
20130101; C12N 2510/00 20130101; C12N 2501/602 20130101; C12N
2740/15043 20130101; C12N 2501/60 20130101; C12N 2501/604
20130101 |
Class at
Publication: |
435/366 ;
435/377 |
International
Class: |
C12N 5/071 20100101
C12N005/071 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 13, 2005 |
JP |
2005-359537 |
Claims
1. A method for preparing an induced pluripotent stem cell, which
comprises: incubating in the presence of basic fibroblast growth
factor a somatic cell in which the following three genes: Oct3/4,
Klf4 and Sox2 are introduced.
2. The method of claim 1, wherein the somatic cell is a human cell.
Description
PRIOR RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/213,035, filed Jun. 13, 2008, which is a
continuation-in-part of PCT/JP2006/324881, filed Dec. 6, 2006,
which claims priority to Japanese Application No. 2005-359537,
filed Dec. 13, 2005, and this application is a continuation of U.S.
patent application Ser. No. 12/213,035, filed Jun. 13, 2008, which
claims priority to U.S. Provisional Application No. 61/001,108,
filed Oct. 31, 2007, and U.S. Provisional Application No.
60/996,289, filed Nov. 9, 2007. The entire disclosures of each of
the above-cited applications are considered as being part of this
application and are expressly incorporated by reference herein in
their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to a nuclear reprogramming
factor having an action of reprogramming a somatic cell to derive
an induced pluripotent stem (iPS) cell. The present invention also
relates to the aforementioned iPS cells, methods of generating and
maintaining iPS cells, and methods of using iPS cells, including
screening and testing methods as well as methods of stem cell
therapy. The present invention also relates to somatic cells
derived by inducing differentiation of the aforementioned iPS
cells.
BACKGROUND OF THE INVENTION
[0003] Embryonic stem cells (ES cells) are stem cells established
from human or mouse early embryos which have a characteristic
feature that they can be cultured over a long period of time while
maintaining pluripotent ability to differentiate into all kinds of
cells existing in living bodies. Human embryonic stem cells are
expected for use as resources for cell transplantation therapies
for various diseases such as Parkinson's disease, juvenile
diabetes, and leukemia, taking advantage of the aforementioned
properties. However, transplantation of ES cells has a problem of
causing rejection in the same manner as organ transplantation.
Moreover, from an ethical viewpoint, there are many dissenting
opinions against the use of ES cells which are established by
destroying human embryos.
[0004] Embryonic stem (ES) cells, derived from the inner cell mass
of mammalian blastocysts, have the ability to grow indefinitely
while maintaining pluripotency (Evans et al., Nature 292:154-156,
1981; Martin, P.N.A.S. USA 78:7634-7638, 1981). These properties
have led to expectations that human ES cells might be useful to
understand disease mechanisms, to screen effective and safe drugs,
and to treat patients of various diseases and injuries, such as
juvenile diabetes and spinal cord injury (Thomson et al., Science
282:1145-1147, 1998). Use of human embryos, however, faces ethical
controversies that hinder the applications of human ES cells. In
addition, it is difficult to generate patient- or disease-specific
ES cells, which are required for their effective application.
Therefore, if dedifferentiation of a patient's own somatic cells
could be induced to establish cells having pluripotency and growth
ability similar to those of ES cells (in this specification, these
cells are referred to as "induced pluripotent stem cells (iPS
cells)", though they are sometimes called "embryonic stem cell-like
cells" or "ES-like cells"), it is anticipated that such cells could
be used as ideal pluripotent cells, free from rejection or ethical
difficulties.
[0005] Methods for nuclear reprogramming of a somatic cell nucleus
have been reported. One technique for nuclear reprogramming which
has been reported involves nuclear transfer into oocytes (Wakayama
et al., Nature 394:369-374, 1998; Wilmut et al., Nature
385:810-813, 1997). Another method, for example, a technique of
establishing an embryonic stem cell from a cloned embryo, prepared
by transplanting a nucleus of a somatic cell into an egg, was
reported (Hwang et al., Science 303:1669-74, 2004; Hwang et al.,
Science 308:1777-83, 2005): these articles were, however, proved to
be fabrications and later withdrawn. Others have reported
techniques for nuclear reprogramming of a somatic cell nucleus by
fusing a somatic cell and an ES cell (Tada et al., Curr. Biol.
11:1553-1558, 2001; Cowan et al., Science 309:1369-73, 2005).
Another reported technique for reprogramming a cell nucleus
involves treatment of a differentiated cell with an
undifferentiated human carcinoma cell extract (Taranger et al.,
Mol. Biol. Cell 16:5719-35, 2005). However, these methods all have
serious drawbacks. Methods of nuclear transfer into oocytes and
techniques which involve the fusion of ES and differentiated cells
both comprise the use of ES cells, which present ethical problems.
In addition, cells generated by such methods often lead to problems
with rejection upon transplantation into an unmatched host.
Furthermore, the use of cell extracts to treat differentiated cells
is technically unreliable and unsafe, in part because the cell
extract components responsible for the nuclear programming are
mixed in solution with other unknown factors.
[0006] A method for screening a nuclear reprogramming factor having
an action of reprogramming differentiated somatic cells to derive
induced pluripotent stems cell was proposed in International
Publication WO2005/80598, which is incorporated by reference in its
entirety. This method comprises the steps of: contacting somatic
cells containing a marker gene under expression regulatory control
of an ECAT (ES cell associated transcript) gene expression control
region with a test substance; examining presence or absence of the
appearance of a cell that expresses the marker gene; and choosing a
test substance inducing the appearance of said cell as a candidate
nuclear reprogramming factor for somatic cells. A method for
reprogramming a somatic cell is disclosed in Example 6 and the like
of the above publication. However, this publication fails to report
an actual identification of a nuclear reprogramming factor.
[0007] In view of these problems, there remains a need in the art
for nuclear reprogramming factors capable of generating pluripotent
stem cells from somatic cells. There also remains a need for
pluripotent stem cells, which can be derived from a patient's own
somatic cells, so as to render ethical issues and avoid problems
with rejection. Such cells would have enormous potential for both
research and clinical applications.
SUMMARY OF THE INVENTION
[0008] The present invention provides induced pluripotent stem
(iPS) cells derived by nuclear reprogramming of a somatic cell. The
present invention also provides methods for reprogramming of a
differentiated cell without using eggs, embryos, or embryonic stem
(ES) cells. The present invention also provides nuclear
reprogramming factors for induction of pluripotent stem cells. The
disclosed methods and nuclear reprogramming factors may be used to
conveniently and highly reproducibly establish iPS cells having
pluripotency and growth ability similar to that of ES cells. More
specifically, the present invention provides for inducing
reprogramming of a differentiated cell without using eggs, embryos,
or ES cells to conveniently and highly reproducibly establish the
iPS cells having pluripotency and growth ability similar to that of
ES cells.
[0009] The invention provides a pluripotent stem cell induced by
reprogramming a somatic cell in the absence of eggs, embryos, or
embryonic stem (ES) cells. The somatic cell can be a mammalian
cell, for example a mouse cell or a human cell. The present
invention also provides such a pluripotent stem cell, wherein the
reprogramming comprises contacting the somatic cell with a nuclear
reprogramming factor.
[0010] The nuclear reprogramming factor can comprise at least one
gene product, for example a protein. The nuclear reprogramming
factor can comprise a gene product of an Oct family gene, a Klf
family gene, a Myc family gene, or a Sox family gene. The nuclear
reprogramming factor can comprise one or more gene products of each
of: an Oct family gene, a Klf family gene, and a Sox family gene.
The nuclear reprogramming factor can comprise one or more gene
products of each of: an Oct family gene, a Klf family gene, a Myc
family gene, and a Sox family gene. Furthermore, the nuclear
reprogramming factor can comprise one or more gene products of each
of: an Oct family gene, a Klf family gene, together with a
cytokine. The cytokine can be at least one of basic fibroblast
growth factor (bFGF) and stem cell factor (SCF).
[0011] The invention also provides a method for preparing an
induced pluripotent stem cell by nuclear reprogramming of a somatic
cell, which comprises contacting a nuclear reprogramming factor
with the somatic cell to obtain an induced pluripotent stem cell.
The invention also provides such a method which is performed in the
absence of eggs, embryos, or embryonic stem (ES) cells. The present
invention also provides an induced pluripotent stem cell obtained
by such a method. The present invention also provides a pluripotent
stem cell induced by reprogramming a somatic cell, wherein the
reprogramming comprises contacting the somatic cell with a nuclear
reprogramming factor.
[0012] The present invention also provides such a method wherein
the nuclear reprogramming factor comprises one or more gene
products of each of: an Oct family gene, a Klf family gene, and a
Sox family gene. The present invention also provides such a method
wherein the nuclear reprogramming factor comprises one or more gene
products of each of Oct3/4, Klf4, and Sox2. The present invention
also provides such a method wherein the nuclear reprogramming
factor further comprises one or more gene products of a Sall4 gene.
The present invention also provides pluripotent stem cells prepared
by such methods.
[0013] The present invention also provides such a method wherein
the nuclear reprogramming factor comprises one or more gene
products of each of: wherein the nuclear reprogramming factor
comprises one or more gene products of each of: an Oct family gene,
a Klf family gene, a Myc family gene, and a Sox family gene. The
present invention also provides such a method wherein the nuclear
reprogramming factor comprises one or more gene products of each
of: Oct3/4, Klf4, c-Myc, and Sox2. The present invention also
provides such a method wherein the nuclear reprogramming factor
further comprises one or more gene products of a Sall4 gene. The
present invention also provides pluripotent stem cells prepared by
such methods.
[0014] The present invention also provides such a method wherein
the nuclear reprogramming factor comprises one or more gene
products of each of: Klf4, c-Myc, Oct3/4, Sox2, Nanog, and Lin28.
The present invention also provides pluripotent stem cells prepared
by such a method.
[0015] The present invention also provides a method of inducing a
somatic cell to become a pluripotent stem cell comprising
contacting the somatic cell with a nuclear reprogramming factor
under conditions to obtain a pluripotent stem cell free of
rejection.
[0016] The present invention also provides a somatic cell derived
by inducing differentiation of an induced pluripotent stem cell as
disclosed herein.
[0017] The present invention also provides a method for stem cell
therapy comprising: (1) isolating and collecting a somatic cell
from a patient; (2) inducing said somatic cell from the patient
into an iPS cell (3) inducing differentiation of said iPS cell, and
(4) transplanting the differentiated cell from (3) into the
patient.
[0018] The present invention also provides a method for evaluating
a physiological function of a compound comprising treating cells
obtained by inducing differentiation of an induced pluripotent stem
cell as disclosed herein with the compound.
[0019] The present invention also provides a method for evaluating
the toxicity of a compound comprising treating cells obtained by
inducing differentiation of an induced pluripotent stem cell as
disclosed herein with the compound.
[0020] Other features and advantages of the present invention will
be set forth in the description of the invention that follows, and
will be apparent, in part, from the description or may be learned
by practice of the invention. The invention will be realized and
attained by the compositions, products, and methods particularly
pointed out in the written description and claims hereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0022] FIG. 1 shows a screening method for reprogramming factors
using embryonic fibroblasts (MEFs) of a mouse having .beta.geo
knock-in Fbx15 gene.
[0023] FIG. 2 depicts photographs showing morphology of iPS cells
obtained by introducing the 24 genes shown in TABLE 4. Morphologies
of differentiated cells (MEF) and of normal embryonic stem cells
(ES) are also shown as a reference.
[0024] FIG. 3 shows expression profiles of marker genes in iPS
cells. The results of RT-PCR using total RNAs extracted from iPS
cells, ES cells and MEF cells as templates are shown.
[0025] FIG. 4 shows methylation status of DNA in iPS cells. Genomic
DNAs extracted from iPS cells, ES cells, and MEF cells were treated
with bisulfite. The target DNAs were amplified by PCR and then
inserted into plasmid. Ten clones of plasmid were isolated for each
of the genes, and sequenced. Methylated CpGs are indicated with
closed circles, and unmethylated CpGs with open circles.
[0026] FIG. 5 shows colony numbers of G418-resistant cells obtained
by transduction of 24-gene group and 23-gene groups wherein each
individual gene was withdrawn from the 24-gene group. The lower
parts of the graph show colony numbers obtained in one week after
the G418 selection, and the upper parts of the graph show numbers
of clones obtained in three weeks. When each boxed gene (the
reference number for each gene is the same as that indicated in
TABLE 4) was withdrawn, no colonies were obtained at all, or only a
few colonies were observed after 3 weeks.
[0027] FIG. 6 shows colony numbers of G418-resistant cells obtained
by transduction of 10-gene group and 9-gene groups wherein each
individual gene was withdrawn from the 10-gene group. When each of
genes #14, #15 or #20 was withdrawn, no colony was obtained. When
gene #22 was withdrawn, a few G418-resistant colonies were
obtained. However, the cells gave differentiated morphology which
was apparently different from that of iPS cells.
[0028] FIG. 7 shows numbers of G418-resistant emerged colonies
(reprogrammed colony) with 10-gene group, 4-gene group, 3-gene
groups, or 2-gene groups. Typical morphology and sizes of the
colonies are shown.
[0029] FIG. 8 depicts photographs showing results of
hematoxylin-eosin (H & E) staining of tumors formed after
subcutaneous transplantation of iPS cells derived from MEFs into
nude mice. Differentiation into a variety of tissues in a
triploblastic system was observed.
[0030] FIG. 9 depicts photographs of embryos prepared by
transplanting iPS cells derived from adult dermal fibroblasts into
mouse blastocysts and transplanting the cells into the uteri of
pseudopregnant mice. It can be observed that, in the upper left
embryo, cells derived from the iPS cells (emitting green
fluorescence) were systemically distributed. In the lower
photographs, it can be observed that almost all cells of the heart,
liver, and spinal cord of the embryo were GFP-positive and were
derived from the iPS cells.
[0031] FIG. 10 depicts photographs showing results of RT-PCR
confirming the expression of the ES cell marker genes. In the
photographs, Sox2 minus indicates iPS cells established by the
transduction of 3 genes into MEFs, 4ECATs indicates iPS cells
established by the transduction of 4 genes into MEFs, 10ECATs
indicates iPS cells established by the transduction of 10 genes
into MEFs, 10ECATs Skin fibroblast indicates iPS cells established
by the transduction of 10 genes into dermal fibroblasts, ES
indicates mouse ES cells, and MEF indicates MEF cells without gene
transduction. The numerical values under the symbols indicate
clones numbers.
[0032] FIG. 11 shows an effect of bFGF on the establishment of iPS
cells from MEFs. Four factors (upper row) or three factors except
for c-Myc (lower row) were retrovirally transduced into MEFs
derived from Fbx15.sup..beta.geo/.beta.geo mice, and cultured on
ordinary feeder cells (STO cells) (left) and bFGF expression
vector-introduced STO cells (right). G418 selection was performed
for 2 weeks, and cells were stained with crystal blue and
photographed. The numerical values indicate the number of
colonies.
[0033] FIGS. 12(A)-(B) depict explanations of the experiments using
Nanog-EGFP-IRES-Puro.sup.r mice. (A) E. coli artificial chromosome
(BAC) containing the mouse Nanog gene in the center was isolated,
and the EGFP-IRES-Puro.sup.r cassette was inserted upstream from
the coding region of Nanog by recombineering. (B) Transgenic mice
were prepared with the modified BAC. GFP expression was observed
limitedly in inner cell masses of blastocysts and gonads.
[0034] FIG. 13 depicts explanations of the experiments using
Nanog-EGFP-IRES-Puro.sup.r mice. From embryos of
Nanog-EGFP-IRES-Puro.sup.r mice (13.5 days after fertilization),
heads, viscera and gonads were removed to establish MEFs. As a
result of analysis with a cell sorter, almost no GFP-positive cells
existed in MEFs derived from the Nanog-EGFP-IRES-Puro mice (Nanog)
in the same manner as the Fbx15.sup..beta.geo/.beta.geo
mouse-derived MEFs (Fbx15) or wild-type mouse-derived MEFs
(Wild).
[0035] FIG. 14 depicts photographs of iPS cells established from
the Nanog-EGFP-IRES-Puro mouse MEFs (left) and the
Fbx15.sup..beta.geo/.beta.geo mouse MEFs (right). The cells were
selected with puromycin and G418, respectively.
[0036] FIG. 15 shows results of growth of iPS cells. 100,000 cells
of each of ES cells, iPS cells derived from the
Nanog-EGFP-IRES-Puro mouse MEFs (Nanog iPS, left), and iPS cells
derived from the Fbx15.sup..beta.geo/.beta.geo mouse MEFs (Fbx iPS)
were seeded on 24-well plates, and passaged every 3 days. Cell
count results are shown. The numerical values represent average
doubling times.
[0037] FIG. 16 shows gene expression profiles of iPS cells.
Expression of the marker genes in MEFs, ES cells, iPS cells derived
from Nanog-EGFP-IRES-Puro mouse MEFs (Nanog iPS, left), and iPS
cells derived from Fbx15.sup..beta.geo/.beta.geo mouse MEFs (Fbx
iPS) were analyzed by RT-PCR. The numerical values at the bottom
indicate the numbers of passages.
[0038] FIG. 17 shows teratoma formation from the Nanog iPS cells.
1,000,000 cells of each of ES cells or Nanog iPS cells #24 (passage
8 times) were subcutaneously injected into the backs of nude mice,
and the appearance of tumors formed after 3 weeks (left) and tissue
images (right, H & E stained) are shown.
[0039] FIG. 18 shows preparation of chimeric mice with the Nanog
iPS cells. The chimeric mice that were born after transplantation
of the Nanog iPS cells (clone NPMF4EK-24, passaged 6 times) into
the blastocysts. Four chimeric mice were born from 90 transplanted
embryos.
[0040] FIG. 19 shows germ-line transmission from the Nanog iPS
cells. PCR analysis of genomic DNA of mice, born by mating of the
chimeric mice shown in FIG. 18 and C57BL/6 mice, revealed the
existence of transgenes of Oct3/4 and Klf4 in all of the mice,
thereby confirming germ-line transmission.
[0041] FIG. 20 shows induction of differentiation into nerve cells
from iPS cells. Nerve cells (top, .beta.III tubulin-positive),
oligodendrocytes (left, O4-positive), and astrocytes (right,
GFAP-positive) differentiated in vitro from dermal
fibroblasts-derived iPS cells are shown.
[0042] FIG. 21 depicts explanations of establishment of the iPS
cells without using drug selection. MEFs at 10,000 to 100,000 cells
per 10 cm dish were seeded, and the 4 factors were retrovirally
transduced. No colony appeared in the control (Mock, top left),
whilst in the dish with the transduction by the 4 factors, swelling
colonies similar to those of the iPS cells were obtained (bottom
left and center), as well as flat transformant colonies. When the
cells were passaged, cells similar to the iPS cells were obtained
(right).
[0043] FIG. 22 shows gene expression profiles of cells established
without using drug selection. RNA was extracted from the
established cells shown in FIG. 21, and expression of the ES cell
marker genes was analyzed by RT-PCR.
[0044] FIG. 23(A)-(B) show iPS cell-like cells derived from human
fibroblasts. The colonies obtained by retroviral transduction with
human homologous genes of the 4 factors into fibroblasts derived
from human embryos (FIG. 23(A)), and the cells after two passages
(FIG. 23(B)) are shown.
[0045] FIG. 24 shows establishment of the iPS cells from human
adult dermal fibroblasts. The factors mentioned in the left column
were transduced retrovirally into human adult dermal fibroblasts
infected with the mouse retroviral receptor with lentivirus. The
photographs shows phase contrast images (object .times.10) on day 8
after the viral infection.
[0046] FIGS. 25(A)-(B) show the results of alkaline phosphatase
staining of iPS cells from two different experiments. (A)
8.times.10.sup.5 HDFs derived from adult skin expressing mouse
Slc7a1 gene and introduced with pMXs encoding the genes indicated
were plated on mitomycin C-treated STO cells. The infected cells
were cultured in ES medium for 12 days. The cells were stained with
alkaline phosphatase. (B) BJ fibroblasts expressing mouse Slc7a1
gene were plated at 8.times.10.sup.5 cells per 100 mm dish on
mitomycin C treated STO cells. Next day, the cells were transduced
with the genes indicated (left) by retroviral infection. After
transduction, the cells were maintained in ES medium for 2 weeks.
After picking up the colonies, the cells were stained with alkaline
phosphatase.
[0047] FIG. 26(A)-(B) show Cyanine 3 (Cy-3) staining of iPS (-like)
cells with ES cell markers. (A) iPS (-like) cells derived from
adult human dermal fibroblasts (HDFs) were plated at
5.times.10.sup.4 cells per well of 6 well plates on mitomycin
C-treated STO cells, and grown for 4 days. The cells were fixed
with PBS containing 10% formalin, and blocked with blocking buffer
(0.1% bovine serum albumin and 10 mg/ml normal donkey serum in PBS)
for 45 minutes at room temperature. Primary antibodies indicated
above were diluted 1:100 in blocking buffer. Overnight incubation
with primary antibody, the cells were washed with PBS, and then
incubated with secondary antibody. Cy-3-conjugated anti-mouse IgG
(for ABCG-2 and SSEA-4) and anti-rat IgM (for SSEA-3) antibodies
were used. (B) iPS (-like) cells derived from adult HDFs (clone
87E3, 87E4 and 87E12) were plated at 5.times.10.sup.4 cells per
well of a 6 well plate on mitomycin C-treated STO cells, and grown
for 5 days. Parental HDFs also plated on 6 well plate and
maintained for 2 days. The cells were fixed with PBS containing 10%
formalin, and blocked with blocking buffer (3% BSA in PBS) for 45
minutes at room temperature. Primary antibodies indicated above
were diluted 1:100 in blocking buffer. Overnight incubation with
primary antibody, the cells were washed with PBS, and then
incubated with secondary antibody. Cy-3-conjugated anti-mouse IgG
(for ABCG-2, E-cadherin, and SSEA-4) and anti-rat IgM (for SSEA-3)
antibodies were used as secondary antibodies.
[0048] FIG. 27 shows human iPS (-like) cells express ECATs. Total
RNA was isolated from human iPS (-like) cells
(iPS-HDFaSlc-87E-1.about.8, 11 and 12), NTERA2 cloneD1 human
embryonic carcinoma cells (passage 35) and adult HDFs expressing
mouse Slc7a1 gene (passage 6). First-strand cDNA was synthesized by
using oligo-dT20 primer and Rever Tra Ace-.alpha.-kit (Toyobo)
according to manufacturer's protocol. PCR was performed with the
primers as follows: hOct4 S1165 and hOct4-AS1283 for endogenous
OCT4, hSox2-S1430 and hSox2-AS1555 for endogenous SOX2,
ECAT4-macaca-9685 and ECAT4-macaca-1334AS for NANOG, hRex1-RT-U and
hRex1-RT-L for REX1, hFGF4-RT-U and hFGF4-RT-L for FGF4, hGDF3-S243
and hGDF3-AS850 for GDF3, hECAT15-S532 and hECAT15-AS916 for
ECAT15-1, hECAT15-2-S85 and hECAT15-2-AS667 for ECAT15-2, hpH34-S40
and hpH34-AS259 for ESG1, hTERT-S3556 and hTERT-AS3713 for hTERT,
and G3PDH-F and G3PDH-R for G3PDH.
[0049] FIG. 28 shows human iPS (-like) cells express ECATs. Total
RNA was isolated from human iPS (-like) cells (iPS-BJSlc-97E-1, 2,
4, 5, 6, 7, 8, 10, 11, 12, -97G-3, 5, -97H-3, 5), NTERA2 clone D1
human embryonic carcinoma cells (passage 35) and BJ fibroblasts
expressing mouse Slc7a1 gene (passage 6). First-strand cDNA was
synthesized by using oligo-dT20 primer and Rever Tra
Ace-.alpha.-kit (Toyobo) according to manufacturer's protocol. PCR
was performed with the primers as follows: hOct4 S1165 and
hOct4-AS1283 for endogenous OCT4, hSox2-S1430 and hSox2-AS1555 for
endogenous SOX2, ECAT4-macaca-968S and ECAT4-macaca-1334AS for
NANOG, hRex1-RT-U and hRex1-RT-L for REX1, hFGF4-RT-U and
hFGF4-RT-L for FGF4, hGDF3-S243 and hGDF3-AS850 for GDF3,
hECAT15-S532 and hECAT15-AS916 for ECAT15-1, hECAT15-2-S85 and
hECAT15-2-AS667 for ECAT15-2, hpH34-S40 and hpH34-AS259 for ESG1,
hTERT-S3556 and hTERT-AS3713 for hTERT, and G3PDH-F and G3PDH-R for
G3PDH.
[0050] FIGS. 29(A)-(D) show teratoma formation. Five million of
hiPS (-like) cells were subcutaneously injected into dorsal flanks
of SCID mouse (female, 5 weeks old). Two months after injection,
large tumors were observed. Tumors were dissected, weighed and
photographed. Then these tumors were fixed with PBS containing 10%
formalin. Paraffin-embedded tumor was sliced and then stained with
hematoxylin and eosin. (A) Mouse from clone iPS-HDFa/Slc-87E-12.
(B)-(D) indicate mouse teratomas from clones iPS-HDFa/Slc-97E-3
(B); iPS-HDFa/Slc-87E-6 (C); and iPS-HDFa/Slc-87E-12 (D).
[0051] FIG. 30 shows in vitro differentiation of human iPS-like
cells. The cells (iPS-HDFaSlc-127F2, E3) were suspended in hES
medium (w/o bFGF). 2.times.10.sup.6 cells were transferred to HEMA
(2-hydroxyethyl methacrylate)-coated 100 mm tissue culture dish.
The medium was changed every other day. After seven days floating
culture, the cells were collected, plated to six gelatinized 35 mm
dishes and incubated another 7 days. The cells were fixed with PBS
containing 10% formalin for 10 min at room temperature,
permeabilized with PBS containing 0.5% TritonX-100 for 5 min at
room temperature, and blocked with PBS containing 3% BSA for 30 min
at room temperature. Primary antibodies used in this experiment
were as follows; anti-.alpha.-smooth muscle actin (Ms mono,
pre-diluted, DAKO), anti-.beta.III-tubulin (Ms mono, 1:100 in
blocking buffer, Chemicon), anti-.alpha.-fetoprotein (Rb poly,
pre-diluted, DAKO), normal mouse IgG (2 mg/ml, Chemicon), and
normal rabbit IgG (2 mg/ml, Chemicon). After incubation with
primary antibody (1 hour at room temperature), the cells were
washed with PBS, and incubated with secondary antibody (1:300 in
blocking buffer).
[0052] FIG. 31 shows improved transduction efficiency of
retroviruses in human HDFs. HDFs or HDFs expressing mouse Slc7a1
gene (HDF-Slc7a1) were introduced with ecotropic (Eco) or
amphotropic (Ampho) pMX retroviruses containing the GFP cDNA. Shown
are results of fluorescent microscope (upper) and flow cytometry
(lower). Bars=100 .mu.m.
[0053] FIGS. 32(A)-(N) show induction of iPS cells from adult HDFs
in primate ES cell media. (A) Time schedule of iPS cell generation.
(B) Morphology of HDFs. (C) Typical image of non-ES cell-like
colony. (D) Typical image of hES cell-like colony. (E) Morphology
of established iPS cell line at passage number 6 (clone 201B7). (F)
Image of iPS cells with high magnification. (G) Spontaneously
differentiated cells in the center part of human iPS cell colonies.
(H-N) Immunocytochemistry for SSEA-1 (H), SSEA-3 (I), SSEA-4 (J),
TRA-1-60 (K), TRA-1-81 (L), TRA-2-4916E (M), and Nanog (N). Nuclei
were stained with Hoechst 33342 (blue). Bars=200 .mu.m (B-E, G), 20
.mu.m (F), and 100 .mu.m (H-N).
[0054] FIGS. 33(A)-(C) show feeder dependency of human iPS cells.
(A) Image of iPS cells plated on gelatin-coated plate. (B) Images
of iPS cells cultured on Matrigel-coated plates in MEF-conditioned
medium (MEF-CCM). (C) Images of iPS cells cultured in ES medium on
Matrigel-coated plates with non-conditioned hES medium.
[0055] FIGS. 34(A)-(E) show expression of hES cell marker genes in
human iPS cells. (A) RT-PCR analysis of ES cell marker genes. (B)
Western blot analysis of ES cell marker genes. (C) Quantitative PCR
for expression of retroviral transgenes. The graph shows the
average of three assays. Bars indicate standard deviation. (D)
Bisulfite genomic sequencing of the promoter regions of OCT3/4,
REXJ and NANOG. Open and closed circles indicate unmethylated and
methylated CpGs. (E) Luciferase assays. The graphs show the average
of the results from four assays. Bars indicate standard
deviation.
[0056] FIGS. 35(A)-(B) show high levels of telomerase activity and
exponential proliferation of human iPS cells. (A) Detection of
telomerase activities by the TRAP method. Heat-inactivated (+)
samples were used as negative controls. IC=internal control. (B)
Growth curve of iPS cells. Shown are averages and standard
deviations in quadruplicate.
[0057] FIGS. 36(A)-(B) show genetic analyses of human iPS cells.
(A) Genomic PCR revealed integration of all the four retroviruses
in all clones. (B) Southern blot analyses with a c-MYC cDNA probe.
Asterisk indicates the endogenous c-MYC alleles (2.7 kb). Arrowhead
indicates mouse c-Myc alleles derived from SNL feeder cells (9.8
kb).
[0058] FIGS. 37(A)-(L) show embryoid body-mediated differentiation
of human iPS cells. (A) Floating culture of iPS cells at day 8.
(B-E) Images of differentiated cells at day 16 (B), neuron-like
cells (C), epithelial cells (D), and cobblestone-like cells (E).
(F-K) Immunocytochemistry of alpha-fetoprotein (F), vimentin (G),
.alpha.-smooth muscle actin (H), desmin (I), .beta.III-tubulin (J),
and GFAP (K). Bars=200 .mu.m (A, B) and 100 .mu.m (C-K). Nuclei
were stained with Hoechst 33342 (blue). (L) RT-PCR analyses of
various differentiation markers for the three germ layers.
[0059] FIGS. 38(A)-(E) show directed differentiations of human iPS
cells. (A) Phase contrast image of differentiated iPS cells after
18 days cultivation on PA6. (B) Immunocytochemistry of the cells
shown in A with .beta.III-tubulin (red) and tyrosine hydroxylase
(green) antibodies. Nuclei were stained with Hoechst 33342 (blue).
(C) RT-PCR analyses of dopaminergic neuron markers. (D) Phase
contrast image of iPS cells differentiated into cardiomyocytes. (E)
RT-PCR analyses of cardiomyocyte markers. Bars=200 .mu.m (A, D) and
100 .mu.m (B).
[0060] FIG. 39 shows hematoxylin and eosin staining of teratoma
derived from human iPS cells (clone 201B7).
[0061] FIG. 40 shows human iPS cells (phase contrast images)
derived from fibroblast-like synoviocytes (HFLS, clone 243111) and
BJ fibroblasts (clone 246G1). Bars=200 .mu.m.
[0062] FIG. 41 shows expression of ES cell marker genes in iPS
cells derived from HFLS and BJ fibroblasts.
[0063] FIG. 42 shows embryoid body-mediated differentiation of iPS
cells derived from HFLS and BJ fibroblasts.
[0064] FIGS. 43(A)-(C) show the effect of family factors and the
omission of Myc on generation of iPS cells from Nanog-reporter
MEFs. (A) Generation of iPS cells with family genes from MEF by
Nanog selection. The number of GFP-positive colonies is shown. The
results of three independent experiments were shown with different
colors (white, gray, and black). The "4 factors" indicate the
combination of Oct3/4, Sox2, Klf4, and c-Myc. (B) The effect of
puromycin selection timing on iPS cell generation. Shown are
GFP-positive colonies observed 28 days after the transduction of
the four factors or the three factors devoid of Myc. (C) The effect
of puromycin selection timing on the percentage of GFP-positive
colonies per all colonies.
[0065] FIG. 44 shows teratomas derived from iPS cells, which were
induced from Fbx15-reporter MEFs with family proteins.
[0066] FIG. 45 shows characterization of iPS cells induced from
Nanog-reporter MEFs without Myc retroviruses. RT-PCR showing
expression levels of ES cell marker genes and the four factors. By
using specific primer sets, total transcripts, transcripts from the
endogenous genes (endo), and the transcripts from the retroviruses
(Tg) were distinguished for the four factors.
[0067] FIGS. 46(A)-(C) show generation of iPS cells without Myc
retroviruses from MEFs containing the Fbx15-reporter and the
constitutively active GFP-transgene. (A) Morphology of iPS cells
generated without Myc retroviruses. The bar indicates 500 .mu.m.
(B) RT-PCR analyses of ES marker genes in ES, MEF, and iPS cells
induced without Myc. (C) Chimeras derived from iPS cells induced
without Myc (clones 142B-6 and -12).
[0068] FIGS. 47(A)-(D) show the efficient isolation of iPS cells
without drug selection. (A) Morphology of iPS cells induced from
adult TTF containing the Nanog-GFP-IRES-Puro.sup.r reporter. Cells
were transduced with either the four factors or the three factors
devoid of Myc, together with DsRed, and then were cultured for 30
days without drug selection. The expression of the Nanog reporter
(Nanog-GFP) and the DsRed retrovirus (Tg-DsRed) was examined by
fluorescent microscopy. The bar indicates 500 .mu.m. (B) Morphology
of iPS cells induced from adult TTF, which contained a DsRed
transgene driven by a constitutively active promoter (ACTB,
.beta.-actin gene), but lacking the Nanog- or Fbx15-selection
cassettes. The cells were transduced with either the four factors
or the three factors devoid of Myc, together with GFP, and then
cultured for 30 days without drug selection. The expression of the
GFP retrovirus (Tg-GFP) was examined by fluorescent microscopy. The
bar indicates 500 .mu.m. (C) RT-PCR analyses of ES maker genes in
iPS cells generated from TTF without drug selection and ES cells.
(D) Chimeras derived from iPS cells, which were generated from
adult TTF without drug selection or the Myc retroviruses.
[0069] FIGS. 48(A)-(C) show induction of human iPS cells without
Myc retroviruses. (A) The retroviruses for Oct3/4, Sox2 and Klf4
were introduced into BJ fibroblasts (246G) or HDF (253G). After 30
days, a few hES cell-like colonies emerged. These cells were
expandable and showed hES cell-like morphology. (B) The expression
of ES cell marker genes in human iPS cells derived from HDF without
Myc retroviruses (253G) or with Myc (253F). (C) Embryoid
body-mediated differentiation of human iPS cells generated without
Myc retroviruses.
[0070] FIG. 49 shows results from experiments using six factors and
two different combinations of four factors. The vertical axis shows
the number of colonies. The term "6F" refers to the six factors
(klf4, c-myc, oct3/4, sox2, nanog and Lin-28), the term "Y4F"
refers to the first combination of four factors (klf4, c-myc,
oct3/4 and sox2), and the term "T4F" refers to the second
combination of four factors (oct3/4, sox2, nanog and Lin-28),
respectively. The term "ES like" refers to ES-like cell colony
morphologically, and the term "total" shows total number of ES-like
cell colonies and non-ES like cell colonies. Exp#1, Exp#2, Exp#3,
and Exp#4 show individual experimental results, respectively.
[0071] FIGS. 50(A)-(C) show a summary of data from experiments
performed with mouse embryonic fibroblasts (MEFs). (A)
1.0.times.10.sup.5 MEF cells obtained from Nanog
GFP.sup.tg/-Fbx15.sup.-/- mouse were seeded on gelatin coated 6
well plates. Next day, four factors (Oct3/4, Klf4, Sox2, c-Myc) or
three factors (Oct3/4, Klf4, Sox2) were retrovirally transduced
into the cells. After 4 days of the infection, cells were re-seeded
1 to 2 or 1 to 6-ratio on 6 well plates covered with mitomycin
C-treated STO cells. Drug selection was started at 14 days or 21
days. At day 28, GFP positive cells were counted and cells were
stained for alkaline phosphatase (AP) and crystal violet (CV). (B)
Summary of the number of the GFP positive colonies from three
independent experiments, Exp. #1, 2, and 3. (C) Percentage of GFP
positive colonies from three independent iPS experiments, Exp. #1,
2, and 3.
[0072] FIG. 51 shows a summary of data from experiments performed
with adult human dermal fibroblasts. 1.0.times.10.sup.5 adult HDF
cells expressing slc7a were seeded on 6 well plates. Next day, four
factors (Oct3/4, Klf4, Sox2, c-Myc) or three factors (Oct3/4, Klf4,
Sox2) were retrovirally transduced into the cells. After 6 days of
the infection, 5.0.times.10.sup.5 cells were re-seeded on 100 mm
plates covered with 1.5.times.10.sup.6 of mitomycin C-treated STO
cells. At day 7 the medium was replaced with Primate ES cell medium
supplemented with 4 ng/ml bFGF. This figure shows colony numbers at
30 days after infections.
DETAILED DESCRIPTION OF THE INVENTION
[0073] Various investigations were conducted to address the
aforementioned need for pluripotent stem cells which can be derived
from a patient's own somatic cells, and for nuclear reprogramming
factors capable of generating pluripotent stem cells from somatic
cells. Investigations were also conducted to identify nuclear
reprogramming factors by using the screening method for a nuclear
reprogramming factor disclosed in International Publication
WO2005/80598. While International Publication WO 2005/80598
discloses a screening method, this document fails to disclose any
nuclear reprogramming factor. Furthermore, this document fails to
specify any nuclear reprogramming factor or candidate nuclear
reprogramming factor which would be capable of generating an
induced pluripotent stem cell.
[0074] Ultimately, 24 kinds of candidate genes were found as genes
relating to nuclear reprogramming, and among them, three kinds of
the genes were found as particularly preferred for nuclear
reprogramming: sometimes these genes are referred to as essential
in certain embodiments. As further discussed throughout the
specification, the nuclear reprogramming factor of the present
invention may contain one or more factors relating to
differentiation, development, proliferation or the like and factors
having other physiological activities, as well as other gene
products which can function as a nuclear reprogramming factor. The
present invention was achieved on the basis of these findings.
[0075] The present invention provides at least the following
advantages and features: induced pluripotent stem (iPS) cells
derived by nuclear reprogramming of a somatic cell, including
methods for reprogramming of a differentiated cell without using
eggs, embryos, or embryonic stem (ES) cells.
[0076] As further discussed herein with respect to the general
guidance for the reprogramming of differentiated cells and the
examples, the present invention also provides various nuclear
reprogramming factors capable of generating pluripotent stem cells
from somatic cells. The nuclear reprogramming factor may comprise
one or more gene products. The nuclear reprogramming factor may
also comprise a combination of gene products. Each nuclear
reprogramming factor may be used alone or in combination with other
nuclear reprogramming factors as disclosed herein. In addition,
nuclear reprogramming may be performed with small molecules,
compounds, or other agents such that iPS cells are obtained.
[0077] In a preferred embodiment, the nuclear reprogramming factor
comprises a gene product of each of the following three kinds of
genes: an Oct family gene, a Klf family gene, and a Sox family
gene. According to a more preferred embodiment of the invention,
there is provided the aforementioned factor comprising a gene
product of each of the following three kinds of genes: Oct3/4,
Klf4, and Sox2.
[0078] In another embodiment of the invention, there is provided a
nuclear reprogramming factor comprising a gene product of each of
the following three kinds of genes: an Oct family gene, a Klf
family gene, and a Myc family gene. According to a preferred
embodiment of the invention, there is provided the aforementioned
factor comprising a gene product of each of the following three
kinds of genes: Oct3/4, Klf4 and c-Myc.
[0079] According to another preferred embodiment, there is provided
the aforementioned factor, which further comprises a gene product
of the following gene: a Sox family gene, and as a more preferred
embodiment, there is provided the aforementioned factor, which
comprises a gene product of Sox2.
[0080] According to still another preferred embodiment, there is
provided the aforementioned factor, which comprises a cytokine
together with the gene product of the Myc family gene, or
alternatively, instead of the gene product of the Myc family gene.
As a more preferred embodiment, there is provided the
aforementioned factor, wherein the cytokine is basic fibroblast
growth factor (bFGF) and/or stem cell factor (SCF). Accordingly, it
is understood that the nuclear reprogramming factor can be with or
without the Myc family gene.
[0081] According to particularly preferred embodiments, there is
provided a nuclear reprogramming factor for a somatic cell, which
comprises a gene product of the TERT gene in addition to a gene
product of each of an Oct family gene, a Klf family gene, a Myc
family gene, and a Sox family gene; and the aforementioned factor,
which comprises a gene product or gene products of one or more
kinds of genes selected from the group consisting of the following
genes: SV40 Large T antigen (SEQ ID NO: 23), HPV16 E6 (SEQ ID NO:
24), HPV16 E7 (SEQ ID NO: 25), and Bmi1, in addition to a gene
product of each of the Oct family gene, the Klf family gene, the
Myc family gene, the Sox family gene, and the TERT gene.
[0082] In addition to these factors, there is provided the
aforementioned factor, which further comprises a gene product or
gene products of one or more kinds of genes selected from the group
consisting of the following: Fbx15, Nanog, ERas, ECAT15-2, Tcl1,
and .beta.-catenin.
[0083] There is also provided the aforementioned factor, which
comprises a gene product or gene products of one or more kinds of
genes selected from the group consisting of the following: ECAT1,
Esg1, Dnmt3L, ECAT8, Gdf3, Sox15, ECAT15-1, Fthl17, Sall4, Rex1,
UTF1, Stella, Stat3, and Grb2.
[0084] The present invention also provides a nuclear reprogramming
factor comprising a gene product or gene products of one or more
kinds of the following genes: Oct3/4, Sox2, Klf4, Nanog, Lin-28,
and c-Myc.
[0085] The present invention also provides a nuclear reprogramming
factor comprising any combination of gene products, small molecules
and/or substances as described herein, further comprising one or
more factors improving the efficiency of iPS cell induction, such
as one or more gene products of a Sall1 or Sall4 gene.
[0086] In another aspect, the present invention provides a method
for preparing an induced pluripotent stem cell by nuclear
reprogramming of a somatic cell, which comprises the step of
contacting the aforementioned nuclear reprogramming factor with the
somatic cell.
[0087] There is also provided the aforementioned method, which
comprises the step of adding the aforementioned nuclear
reprogramming factor to a culture of the somatic cell; the
aforementioned method, which comprises the step of introducing a
gene encoding the aforementioned nuclear reprogramming factor into
the somatic cell; the aforementioned method, which comprises the
step of introducing said gene into the somatic cell by using a
recombinant vector containing at least one kind of gene encoding
the aforementioned nuclear reprogramming factor; and the
aforementioned method, wherein a somatic cell isolated from a
patient is used as the somatic cell.
[0088] In another aspect, the present invention provides an induced
pluripotent stem cell obtained by the aforementioned method. The
present invention also provides a somatic cell derived by inducing
differentiation of the aforementioned induced pluripotent stem
cell.
[0089] The present invention further provides a method for stem
cell therapy, which comprises the step of transplanting a somatic
cell, wherein said cell is obtained by inducing differentiation of
an induced pluripotent stem cell obtained by the aforementioned
method using a somatic cell isolated and collected from a patient,
into said patient. Several kinds of, preferably approximately 200
kinds of iPS cells prepared from somatic cells derived from healthy
humans can be stored in an iPS cell bank as a library of iPS cells,
and one kind or more kinds of the iPS cells in the library can be
used for preparation of somatic cells, tissues, or organs that are
free of rejection by a patient to be subjected to stem cell
therapy.
[0090] The present invention further provides a method for
evaluating a physiological function or toxicity of a compound, a
medicament, a poison or the like by using various cells obtained by
inducing differentiation of an induced pluripotent stem cell
obtained by the aforementioned method.
[0091] The present invention also provides a method for improving
ability of differentiation and/or growth of a cell, which comprises
the step of contacting the aforementioned nuclear reprogramming
factor with the cell, and further provides a cell obtained by the
aforementioned method, and a somatic cell derived by inducing
differentiation of a cell obtained by the aforementioned
method.
[0092] By using the nuclear reprogramming factor provided by the
present invention, reprogramming of a differentiated cell nucleus
can be conveniently and highly reproducibly induced without using
embryos or ES cells, and an induced pluripotent stem cell, as an
undifferentiated cell having differentiation ability, pluripotency,
and growth ability similar to those of ES cells, can be
established. For example, an induced pluripotent stem cell having
high growth ability and differentiation pluripotency can be
prepared from a patient's own somatic cell by using the nuclear
reprogramming factor of the present invention. Cells obtainable by
differentiating said cell (for example, cardiac muscle cells,
insulin producing cells, nerve cells and the like) are extremely
useful, because they can be utilized for stem cell transplantation
therapies for a variety of diseases such as cardiac insufficiency,
insulin dependent diabetes mellitus, Parkinson's disease and spinal
cord injury, thereby the ethical problem concerning the use of
human embryo and rejection after transplantation can be avoided.
Further, various cells obtainable by differentiating the induced
pluripotent stem cell (for example, cardiac muscle cells, hepatic
cells and the like) are highly useful as systems for evaluating
efficacy or toxicity of compounds, medicaments, poisons and the
like.
[0093] As noted above, transplantation of ES cells has a problem of
causing rejection in the same manner as organ transplantation.
Moreover, from an ethical viewpoint, there are many dissenting
opinions against the use of ES cells, which are established by
destroying human embryos.
[0094] The present invention provides at least the following
advantages and features:
[0095] Identification of Nuclear Reprogramming Factors
[0096] As will be further disclosed below, the nuclear
reprogramming factor of the present invention may contain one or
more factors relating to differentiation, development,
proliferation or the like and factors having other physiological
activities, as well as other gene products which can function as a
nuclear reprogramming factor. It is understood that such
embodiments fall within the scope of the present invention, and the
present invention is, in other words, directed to factors inducing
pluripotent stem cells and various methods of obtaining induced
pluripotent stem cells, including various manners of reprogramming
differentiated cells as well as various manners of culturing,
maintaining, and differentiating the induced pluripotent stem
cells.
[0097] Furthermore, by using somatic cells in which only one or two
genes among the three kinds of genes Oct3/4, Klf4, and c-Myc are
expressed, other gene products which can function as a nuclear
reprogramming factor can be identified by, for example, performing
screening for a gene product which can induce nuclear reprogramming
of said cells. For example, depending on the kinds of genes
expressed in a differentiated cell, one or more genes useful as a
reprogramming factor can be determined using the guidance herein
provided. According to the present invention, the aforementioned
screening method is also provided as a novel method for screening
for a nuclear reprogramming factor. In other words, the present
invention is not limited to any particular combination of nuclear
reprogramming factors and the nuclear reprogramming factors of the
present invention can be identified by screening methods, for
example, the aforementioned screening method.
[0098] In one embodiment, the nuclear reprogramming factor of the
present invention is characterized in that it comprises one or more
gene products. As a means for confirming the nuclear reprogramming
factor of the present invention, for example, the screening method
for nuclear reprogramming factors disclosed in International
Publication WO 2005/80598 can be used. The entire disclosure of the
aforementioned publication is incorporated into the disclosure of
the specification by reference. By referring to the aforementioned
publication, those skilled in the art can perform screening of
nuclear reprogramming factors to confirm the existence and the
action of the reprogramming factor of the present invention.
[0099] For example, as an experimental system allowing for
observation of the reprogramming phenomenon, a mouse can be used in
which the .beta.geo (a fusion gene of the .beta. galactosidase gene
and the neomycin resistance gene) is knocked into the Fbx15 locus
(Tokuzawa et al., Mol. Cell Biol. 23:2699-708, 2003). The details
are described in the examples of the specification. The mouse Fbx15
gene is a gene specifically expressed in differentiation
pluripotent cells such as ES cells and early embryos. In a
homomutant mouse in which .beta.geo is knocked into the mouse Fbx15
gene so as to be deficient in the Fbx15 function, abnormal
phenotypes including those relating to differentiation pluripotency
or generation are not generally observed. In this mouse, the
expression of the .beta.geo is controlled by the enhancer and
promoter of the Fbx15 gene, and differentiated somatic cells in
which .beta.geo is not expressed have sensitivity to G418. In
contrast, .beta.geo knockin homomutant ES cells have resistance
against G418 at an extremely high concentration (higher than 12
mg/ml). By utilizing this phenomenon, an experimental system can be
constructed to visualize reprogramming of somatic cells.
[0100] By applying the aforementioned experimental system,
fibroblasts (Fbx15.sup..beta.geo/.beta.geo MEFs) can be first
isolated from an embryo of the .beta.geo knockin homomutant mouse
(13.5 days after fertilization). The MEFs do not express the Fbx15
gene, and accordingly also do not express .beta.geo to give
sensitivity to G418. However, when the MEFs are fused with genetic
manipulation-free ES cells (also have sensitivity to G418),
.beta.geo is expressed and the cells become G418-resistant as a
result of reprogramming of nuclei of MEFs. Therefore, by utilizing
this experimental system, the reprogramming phenomenon can be
visualized as G418 resistance.
[0101] Nuclear reprogramming factors can be selected by using the
aforementioned experimental system. As candidates of genes relevant
to nuclear reprogramming factors, a plurality of genes can be
selected which show specific expression in ES cells or of which
important roles in the maintenance of pluripotency of ES cells are
suggested, and it can be confirmed whether or not each candidate
gene can induce nuclear reprogramming alone or in an appropriate
combination thereof. For example, a combination of all of the
selected primary candidate genes is confirmed to be capable of
inducing the reprogramming of differentiated cells into a state
close to that of ES cells. Combinations are then prepared by
withdrawing each individual gene from the aforementioned
combination, and the same actions of the combinations are confirmed
in order to select each secondary candidate gene whose absence
causes a reduction of the reprogramming induction ability or loss
of the reprogramming induction ability. By repeating similar steps
for the secondary candidate genes selected as described above, an
essential combination of nuclear reprogramming genes can be
selected, and it can be confirmed that a combination of gene
products of each of the three kinds of genes, e.g., an Oct family
gene, a Klf family gene, and a Myc family gene, acts as a nuclear
reprogramming factor. It can be further confirmed that a
combination of a gene product of a Sox family gene additionally
with the gene products of the aforementioned three kinds of genes
has extremely superior characteristics as a nuclear reprogramming
factor. Specific examples of the selection method for the nuclear
reprogramming factors are demonstrated in the examples of the
specification.
[0102] Therefore, by referring to the above general explanations
and specific explanations of the examples, those skilled in the art
can readily confirm that the combination of these three kinds of
genes induces the reprogramming of somatic cells, and that the
combination of these three kinds of gene products is essential for
nuclear reprogramming in certain embodiments. Thus, the embodiments
herein illustrate various combinations of gene products and/or
nuclear reprogramming factors which can provide iPS cells. In other
words, based on the disclosure provided herein, one of ordinary
skill in the art would know from the disclosed examples and/or
readily determine which combination and/or combinations of nuclear
reprogramming factors, including gene products, can generate
pluripotent stem cells.
[0103] Nuclear Reprogramming Factor (NRF)
[0104] In a preferred embodiment, the NRF comprises a gene product.
The nuclear reprogramming factor can be used to induce
reprogramming of a differentiated cell without using eggs, embryos,
or ES cells, to conveniently and highly reproducibly establish an
induced pluripotent stem cell having pluripotency and growth
ability similar to those of ES cells. For example, the nuclear
reprogramming factor can be introduced into a cell by transducing
the cell with a recombinant vector comprising a gene encoding the
nuclear reprogramming factor. Accordingly, the cell can express the
nuclear reprogramming factor expressed as a product of a gene
contained in the recombinant vector, thereby inducing reprogramming
of a differentiated cell.
[0105] The nuclear reprogramming factor may comprise a protein or
peptide. The protein may be produced from the aforementioned gene,
or alternatively, in the form of a fusion gene product of said
protein with another protein, peptide or the like. The protein or
peptide may be a fluorescent protein and/or a fusion protein. For
example, a fusion protein with green fluorescence protein (GFP) or
a fusion gene product with a peptide such as a histidine tag can
also be used. Further, by preparing and using a fusion protein with
the TAT peptide derived form the virus HIV, intracellular uptake of
the nuclear reprogramming factor through cell membranes can be
promoted, thereby enabling induction of reprogramming only by
adding the fusion protein to a medium thus avoiding complicated
operations such as gene transduction. Since preparation methods of
such fusion gene products are well known to those skilled in the
art, skilled artisans can easily design and prepare an appropriate
fusion gene product depending on the purpose.
[0106] Nuclear reprogramming may also be accomplished with one or
more small molecules, compounds, including inorganic and organic
compounds, or mixtures thereof, extracts, epigenetic factors,
and/or other components of the cytoplasm of a pluripotent cell.
[0107] In a particularly preferred embodiment, the nuclear
reprogramming factor may comprise one or more gene products of each
of the following three kinds of genes: an Oct family gene, a Klf
family gene, and a Sox family gene.
[0108] In another preferred embodiment, the nuclear reprogramming
factor may comprise one or more gene products of each of: an Oct
family gene, a Klf family gene, and a Myc family gene.
[0109] The nuclear reprogramming factor may also comprise one or
more gene products of each of: an Oct family gene, a Klf family
gene, a Myc family gene, and a Sox family gene.
[0110] The nuclear reprogramming factor may also comprise one or
more gene products of each of: an Oct family gene, a Klf family
gene, and a cytokine. In one exemplary embodiment, the
above-referenced nuclear reprogramming factor may further comprise
one or more gene products of a Myc family gene. In another
exemplary embodiment, the above referenced nuclear reprogramming
factor may further comprise one or more gene products of a Sox
family gene.
[0111] The cytokines of the present invention are not particularly
limited. For example, the cytokine may comprise basic fibroblast
growth factor (bFGF/FGF2) or stem cell factor (SCF).
[0112] With regard to gene family members, the nuclear
reprogramming factor may comprise any combination of members from
one or more gene families. For example, a combination of one or
more gene products of Oct3/4, Klf4, and c-Myc. Examples of the Oct
family gene include, for example, Oct3/4, Oct 1A, Oct6, and the
like. Oct3/4 is a transcription factor belonging to the POU family,
and is reported as a marker of undifferentiated cells (Okamoto et
al., Cell 60:461-72, 1990). Oct3/4 is also reported to participate
in the maintenance of pluripotency (Nichols et al., Cell 95:379-91,
1998). Examples of the Klf family gene include Klf1, Klf2, Klf4,
Klf5 and the like. Klf4 (Kruppel like factor-4) is reported as a
tumor repressing factor (Ghaleb et al., Cell Res. 15:92-96, 2005).
Examples of the Myc family gene include c-Myc, N-Myc, L-Myc and the
like. c-Myc is a transcription control factor involved in
differentiation and proliferation of cells (Adhikary & Eilers,
Nat. Rev. Mol. Cell Biol. 6:635-45, 2005), and is also reported to
be involved in the maintenance of pluripotency (Cartwright et al.,
Development 132:885-96, 2005). The NCBI accession numbers of the
genes of the families other than Oct3/4, Klf4 and c-Myc are set in
TABLE 1 as follows:
TABLE-US-00001 TABLE 1 Mouse Human Klf1 Kruppel-like factor 1
(erythroid) NM_010635 NM_006563 Klf2 Kruppel-like factor 2 (lung)
NM_008452 NM_016270 Klf5 Kruppel-like factor 5 NM_009769 NM_001730
c-Myc myelocytomatosis oncogene NM_010849 NM_002467 N-Myc v-Myc
myelocytomatosis viral related NM_008709 NM_005378 oncogene,
neuroblastoma derived (avian) L-Myc v-Myc myelocytomatosis viral
oncogene NM_008506 NM_005376 homolog 1, lung carcinoma derived
(avian) Oct1A POU domain, class 2, transcription factor 1 NM_198934
NM_002697 Oct6 POU domain, class 3, transcription factor 1
NM_011141 NM_002699
[0113] All of these genes are those commonly existing in mammals
including human, and for use of the aforementioned gene products in
the present invention, genes derived from arbitrary mammals (those
derived from mammals such as mouse, rat, bovine, ovine, horse, and
ape) can be used. In addition to wild-type gene products, mutant
gene products including substitution, insertion, and/or deletion of
several (for example, 1 to 10, preferably 1 to 6, more preferably 1
to 4, still more preferably 1 to 3, and most preferably 1 or 2)
amino acids and having similar function to that of the wild-type
gene products can also be used. For example, as a gene product of
c-Myc, a stable type product (T58A) may be used as well as the
wild-type product. The above explanation may be applied similarly
to the other gene products.
[0114] The nuclear reprogramming factor of the present invention
may comprise a gene product other than the aforementioned three
kinds of gene products. An example of such gene product includes a
gene product of a Sox family gene. Examples of the Sox family genes
include, for example, Sox1, Sox3, Sox7, Sox15, Sox17 and Sox18, and
a preferred example includes Sox2. A nuclear reprogramming factor
comprising at least a combination of the gene products of four
kinds of genes, an Oct family gene (for example, Oct3/4), a Klf
family gene (for example, Klf4), a Myc family gene (for example,
c-Myc), and a Sox family gene (for example, Sox2) is a preferred
embodiment of the present invention from a viewpoint of
reprogramming efficiency, and in particular, a combination of a
gene product of a Sox family gene is sometimes preferred to obtain
pluripotency. Sox2, expressed in an early development process, is a
gene encoding a transcription factor (Avilion et al., Genes Dev.
17:126-40, 2003). The NCBI accession numbers of Sox family genes
other than Sox2 are in TABLE 2 as follows.
TABLE-US-00002 TABLE 2 Mouse Human Sox1 SRY-box containing gene 1
NM_009233 NM_005986 Sox3 SRY-box containing gene 3 NM_009237
NM_005634 Sox7 SRY-box containing gene 7 NM_011446 NM_031439 Sox15
SRY-box containing gene 15 NM_009235 NM_006942 Sox17 SRY-box
containing gene 17 NM_011441 NM_022454 Sox18 SRY-box containing
gene 18 NM_009236 NM_018419
[0115] Further, a gene product of a Myc family gene may be replaced
with a cytokine. As the cytokine, for example, SCF, bFGF or the
like is preferred. However, cytokines are not limited to these
examples.
[0116] As a more preferred embodiment, an example includes a factor
which induces immortalization of cells, in addition to the
aforementioned three kinds of gene products, preferably, the four
kinds of gene products. For example, an example includes a
combination of a factor comprising a gene product of the TERT gene.
In another exemplary embodiment, the nuclear reprogramming factor
comprises any of the aforementioned gene products in combination
with a factor comprising a gene product or gene products of one or
more kinds of the following genes: SV40 Large T antigen, HPV16 E6,
HPV16 E7, and Bmi1. TERT is essential for the maintenance of the
telomere structure at the end of chromosome at the time of DNA
replication, and the gene is expressed in stem cells or tumor cells
in humans, whilst it is not expressed in many somatic cells
(Horikawa et al., P.N.A.S. USA 102:18437-442, 2005). SV40 Large T
antigen, HPV16 E6, HPV16 E7, or Bmi1 was reported to induce
immortalization of human somatic cells in combination with Large T
antigen (Akimov et al., Stem Cells 23:1423-33, 2005; Salmon et al.,
Mol. Ther. 2:404-14, 2000). These factors are extremely useful
particularly when iPS cells are induced from human cells. The NCBI
accession numbers of TERT and Bmi1 genes are listed in TABLE 3 as
follows.
TABLE-US-00003 TABLE 3 Mouse Human TERT telomerase reverse
transcriptase NM_009354 NM_198253 Bmi1 B lymphoma Mo-MLV insertion
NM_007552 NM_005180 region 1
[0117] Furthermore, a gene product or gene products of one or more
kinds of genes selected from the group consisting of the following:
Fbx15, Nanog, ERas, ECAT15-2, Tcl1, and .beta.-catenin may be
combined. As a particularly preferred embodiment from a viewpoint
of reprogramming efficiency, an example includes a nuclear
reprogramming factor comprising a total of ten kinds of gene
products, wherein gene products of Fbx15, Nanog, ERas, ECAT15-2,
Tcl1, and .beta.-catenin are combined with the aforementioned four
kinds of gene products. Fbx15 (Tokuzawa et al., Mol. Cell Biol.
23:2699-708, 2003), Nanog (Mitsui et al., Cell 113:631-42, 2003),
ERas (Takahashi et al. Nature 423:541-45, 2003), and ECAT15-2
(Bortvin et al., Development 130:1673-80, 2003) are genes
specifically expressed in ES cells. Tcl1 is involved in the
activation of Akt (Bortvin et al., Development 130:1673-80, 2003),
and .beta.-catenin is an important factor constituting the Wnt
signal transmission pathway, and also reported to be involved in
the maintenance of pluripotency (Sato et al, Nat. Med. 10:55-63,
2004).
[0118] Further, the nuclear reprogramming factor of the present
invention may comprise, for example, a gene product or gene
products of one or more kinds of genes selected from the group
consisting of the following: ECAT1, Esg1, Dnmt3L, ECAT8, Gdf3,
Sox15, ECAT15-1, Fthl17, Sall4, Rex1, UTF1, Stella, Stat3, and
Grb2. ECAT1, Esg1, ECAT8, Gdf3, and ECAT15-1 are genes specifically
expressed in ES cells (Mitsui et al., Cell 113:631-42, 2003).
Dnmt3L is a DNA methylating enzyme-related factor, and Sox15 is a
class of genes expressed in an early development process and
encoding transcription factors (Maruyama et al., J. Biol. Chem.
280:24371-79, 2005). Fthl17 encodes ferritin heavy polypeptide-like
17 (colLoriot et al., Int. J. Cancer 105:371-76, 2003), Sall4
encodes a Zn finger protein abundantly expressed in embryonic stem
cells (Kohlhase et al., Cytogenet. Genome Res. 98:274-77, 2002),
and Rex1 encodes a transcription factor locating downstream from
Oct3/4 (Ben-Shushan et al., Mol. Cell Biol. 18:1866-78, 1998). UTF1
is a transcription cofactor locating downstream from Oct3/4, and it
is reported that the suppression of the proliferation of ES cells
is induced when this factor is suppressed (Okuda et al., EMBO J.
17:2019-32, 1998). Stat3 is a signal factor for proliferation and
differentiation of cells. The activation of Stat3 triggers the
operation of LIF, and thereby the factor plays an important role
for the maintenance of pluripotency (Niwa et al., Genes Dev.
12:2048-60, 1998). Grb2 encodes a protein mediating between various
growth factor receptors existing in cell membranes and the Ras/MAPK
cascade (Cheng et al. Cell 95:793-803, 1998).
[0119] However, as noted above, the gene products which may be
included in the nuclear reprogramming factor of the present
invention are not limited to the gene products of the genes
specifically explained above. The nuclear reprogramming factor of
the present invention may contain one or more factors relating to
differentiation, development, proliferation or the like and factors
having other physiological activities, as well as other gene
products which can function as a nuclear reprogramming factor. It
is understood that such embodiments fall within the scope of the
present invention. By using somatic cells in which only one or two
genes among the three kinds of the gene Oct3/4, Klf4, and c-Myc are
expressed, the other gene products which can function as a nuclear
reprogramming factor can be identified by, for example, performing
screening for a gene product which can induce nuclear reprogramming
of said cells. According to the present invention, the
aforementioned screening method is also provided as a novel method
for screening for a nuclear reprogramming factor.
[0120] Cells of the Invention and Methods of Generating the
Same
[0121] By using the nuclear reprogramming factor of the present
invention, the nucleus of a somatic cell can be reprogrammed to
obtain an induced pluripotent stem cell. In the specification, the
term "induced pluripotent stem cells" means cells having properties
similar to those of ES cells, and more specifically, the term
encompasses undifferentiated cells having pluripotency and growth
ability. However, the term should not be construed narrowly in any
sense, and should be construed in the broadest sense. The method
for preparing induced pluripotent stem cells by using a nuclear
reprogramming factor is explained in International Publication
WO2005/80598 (the term "ES-like cells" is used in the publication),
and a means for isolating induced pluripotent stem cells is also
specifically explained. Therefore, by referring to the
aforementioned publication, those skilled in the art can easily
prepare induced pluripotent stem cells by using the nuclear
reprogramming factor of the present invention. Methods for
preparing induced pluripotent stem cells from somatic cells by
using the nuclear reprogramming factor of the present invention are
not particularly limited. Any method may be employed as long as the
nuclear reprogramming factor can contact with somatic cells under
an environment in which the somatic cells and induced pluripotent
stem cells can proliferate. An advantage of the present invention
is that an induced pluripotent stem cell can be prepared by
contacting a nuclear reprogramming factor with a somatic cell in
the absence of eggs, embryos, or embryonic stem (ES) cells.
[0122] For example, a gene product contained in the nuclear
reprogramming factor of the present invention may be added to a
medium. Alternatively, by using a vector containing a gene that is
capable of expressing the nuclear reprogramming factor of the
present invention, a means of transducing said gene into a somatic
cell may be employed. When such vector is used, two or more kinds
of genes may be incorporated into the vector, and each of the gene
products may be simultaneously expressed in a somatic cell. When
one or more of the gene products contained in the nuclear
reprogramming factor of the present invention are already expressed
in a somatic cell to be reprogrammed, said gene products may be
excluded from the nuclear reprogramming factor of the present
invention. It is understood that such embodiments fall within the
scope of the present invention.
[0123] As indicated above, the nuclear reprogramming factor of the
present invention can be used to generate iPS cells from
differentiated adult somatic cells. In the preparation of induced
pluripotent stem cells by using the nuclear reprogramming factor of
the present invention, types of somatic cells to be reprogrammed
are not particularly limited, and any kind of somatic cells may be
used. For example, matured somatic cells may be used, as well as
somatic cells of an embryonic period. Other examples of cells
capable of being generated into iPS cells and/or encompassed by the
present invention include mammalian cells such as fibroblasts, B
cells, T cells, dendritic cells, ketatinocytes, adipose cells,
epithelial cells, epidermal cells, chondrocytes, cumulus cells,
neural cells, glial cells, astrocytes, cardiac cells, esophageal
cells, muscle cells, melanocytes, hematopoietic cells, pancreatic
cells, hepatocytes, macrophages, monocytes, mononuclear cells, and
gastric cells, including gastric epithelial cells. The cells can be
embryonic, or adult somatic cells, differentiated cells, cells with
an intact nuclear membrane, non-dividing cells, quiescent cells,
terminally differentiated primary cells, and the like.
[0124] Induced pluripotent stem cells may express any number of
pluripotent cell markers, including: alkaline phosphatase (AP);
ABCG2; stage specific embryonic antigen-1 (SSEA-1); SSEA-3; SSEA-4;
TRA-1-60; TRA-1-81; Tra-2-49/6E; ERas/ECAT5, E-cadherin;
.beta.III-tubulin; .alpha.-smooth muscle actin (.alpha.-SMA);
fibroblast growth factor 4 (Fgf4), Cripto, Dax1; zinc finger
protein 296 (Zfp296); N-acetyltransferase-1 (Nat1); (ES cell
associated transcript 1 (ECAT1); ESG1/DPPA5/ECAT2; ECAT3; ECAT6;
ECAT7; ECAT8; ECAT9; ECAT10; ECAT15-1; ECAT15-2; Fthl17; Sall4;
undifferentiated embryonic cell transcription factor (Utf1); Rex 1;
p53; G3PDH; telomerase, including TERT; silent X chromosome genes;
Dnmt3a; Dnmt3b; TRIM28; F-box containing protein 15 (Fbx15);
Nanog/ECAT4; Oct3/4; Sox2; Klf4; c-Myc; Esrrb; TDGF1; GABRB3;
Zfp42, FoxD3; GDF3; CYP25A1; developmental pluripotency-associated
2 (DPPA2); T-cell lymphoma breakpoint 1 (Tcl1); DPPA3/Stella;
DPPA4; other general markers for pluripotency, etc. Other markers
can include Dnmt3L; Sox15; Stat3; Grb2; SV40 Large T Antigen; HPV16
E6; HPV16 E7, .beta.-catenin, and Bmi1. Such cells can also be
characterized by the down-regulation of markers characteristic of
the differentiated cell from which the iPS cell is induced. For
example, iPS cells derived from fibroblasts may be characterized by
down-regulation of the fibroblast cell marker Thy1 and/or
up-regulation of SSEA-1. It is understood that the present
invention is not limited to those markers listed herein, and
encompasses markers such as cell surface markers, antigens, and
other gene products including ESTs, RNA (including microRNAs and
antisense RNA), DNA (including genes and cDNAs), and portions
thereof.
[0125] When induced pluripotent stem cells are used for therapeutic
treatment of diseases, it is desirable to use somatic cells
isolated from patients. For example, somatic cells involved in
diseases, somatic cells participating in therapeutic treatment of
diseases and the like can be used. A method for selecting induced
pluripotent stem cells that appear in a medium according to the
method of the present invention is not particularly limited, and a
well-known means may be suitably employed, for example, a drug
resistance gene or the like can be used as a marker gene to isolate
induced pluripotent stem cells using drug resistance as an index.
Various media that can maintain undifferentiated state and
pluripotency of ES cells and various media which cannot maintain
such properties are known in this field, and induced pluripotent
stem cells can be efficiently isolated by using a combination of
appropriate media. Differentiation and proliferation abilities of
isolated induced pluripotent stem cells can be easily confirmed by
those skilled in the art by using confirmation means widely applied
to ES cells.
[0126] Thus, another preferred embodiment of the invention
comprises a pluripotent stem cell induced by reprogramming a
somatic cell in the absence of eggs, embryos, or embryonic stem
(ES) cells. The pluripotent stem cell can be a mammalian cell, for
example a mouse, human, rat, bovine, ovine, horse, hamster, dog,
guinea pig, or ape cell. For example, direct reprogramming of
somatic cells provides an opportunity to generate patient- or
disease-specific pluripotent stem cells. Mouse iPS cells are
indistinguishable from ES cells in morphology, proliferation, gene
expression, and teratoma formation. Furthermore, when transplanted
into blastocysts, mouse iPS cells can give rise to adult chimeras,
which are competent for germline transmission (Maherali et al.,
Cell Stem Cell 1:55-70, 2007; Okita et al., Nature 448:313-17,
2007; Wernig et al., Nature 448:318-324, 2007). Human iPS cells are
also expandable and indistinguishable from human embryonic stem
(ES) cells in morphology and proliferation. Furthermore, these
cells can differentiate into cell types of the three germ layers in
vitro and in teratomas.
[0127] The present invention also provides for the generation of
somatic cells derived by inducing differentiation of the
aforementioned pluripotent stem cells. The present invention thus
provides a somatic cell derived by inducing differentiation of the
aforementioned induced pluripotent stem cell.
[0128] In another embodiment, there is disclosed a method for
improving differentiation ability and/or growth ability of a cell,
which comprises contacting a nuclear reprogramming factor with a
cell.
[0129] In a particularly preferred embodiment, the present
invention comprises a method for stem cell therapy comprising: (1)
isolating and collecting a somatic cell from a patient; (2)
inducing said somatic cell from the patient into an iPS cell; (3)
inducing differentiation of said iPS cell, and (4) transplanting
the differentiated cell from step (3) into the patient.
[0130] In another preferred embodiment, the present invention
includes a method for evaluating a physiological function of a
compound comprising treating cells obtained by inducing
differentiation of an induced pluripotent stem cell with the
compound.
[0131] A method for evaluating the toxicity of a compound
comprising treating cells obtained by inducing differentiation of
an induced pluripotent stem cell in the presence of the
compound.
EXAMPLES
[0132] Various terms, abbreviations and designations for the raw
materials and tests used in the following Examples are explained as
follows:
Abbreviations
[0133] iPS cell (induced pluripotent stem cell)
[0134] NRF (nuclear reprogramming factor)
[0135] ES cell (embryonic stem cell)
[0136] TTF (tail tip fibroblast)
[0137] MEF (mouse embryonic fibroblast)
[0138] HDF (human dermal fibroblast)
[0139] bFGF (basic fibroblast growth factor)
[0140] SCF (stem cell factor)
[0141] GFP (green fluorescent protein)
[0142] The present invention will be more specifically explained
with reference to examples. However, the scope of the present
invention is not limited to the following examples.
Example 1
Selection of a Nuclear Reprogramming Factor
[0143] In order to identify reprogramming factors, an experimental
system for easy observation of the reprogramming phenomenon is
required. As an experimental system, a mouse in which .beta.geo (a
fusion gene of .beta.-galactosidase gene and neomycin resistance
gene) was knocked into the Fbx15 locus (Tokuzawa et al., Mol. Cell
Biol. 23:2699-708, 2003) was used. The mouse Fbx15 gene is a gene
specifically expressed in differentiation pluripotent cells such as
ES cells and early embryos. However, in a homomutant mouse in which
.beta.geo was knocked into the mouse Fbx15 gene so as to delete the
function of Fbx15, no abnormal phenotypes including those
concerning differentiation pluripotency or development were
observed. In this mouse, expression control of .beta.geo is
attained by the enhancer and promoter of the Fbx15 gene.
Specifically, .beta.geo is not expressed in differentiated somatic
cells, and they have sensitivity to G418. In contrast, the
.beta.geo knockin homomutant ES cells have resistance against G418
at an extremely high concentration (higher than 12 mg/ml). By
utilizing the above phenomenon, an experimental system for
visualizing the reprogramming of somatic cells was constructed.
[0144] In the aforementioned experimental system, fibroblasts
(Fbx15.sup..beta.geo/.beta.geo MEFs) were first isolated from an
embryo of the .beta.geo knockin homomutant mouse (13.5 days after
fertilization). Since MEFs do not express the Fbx15 gene, the cells
also do not express .beta.geo and thus have sensitivity to G418.
Whilst when the MEFs are fused with ES cells that have not been
gene-manipulated (also having sensitivity to G418), the nuclei of
MEFs are reprogrammed, and as a result, .beta.geo is expressed to
give G418-resistance. The reprogramming phenomenon can thus be
visualized as G418 resistance by using this experimental system
(International Publication WO2005/80598). Searches for
reprogramming factors were performed by using the aforementioned
experimental system (FIG. 1), and total 24 kinds of genes were
selected as candidate reprogramming factors, including genes
showing specific expression in ES cells and genes suggested to have
important roles in the maintenance of differentiation pluripotency
of ES cells. These genes are shown in TABLES 4 and 5 below. For
.beta.-catenin (#21) and c-Myc (#22), active type mutants (catenin:
S33Y, c-Myc: T58A) were used.
TABLE-US-00004 TABLE 4 Number Name of Gene Explanation of Gene 1
ECAT1 ES cell associated transcript 1 (ECAT1) 2 ECAT2 developmental
pluripotency associated 5 (DPPA5), ES cell specific gene 1 (ESG1) 3
ECAT3 F-box protein 15 (Fbx15), 4 ECAT4 homeobox transcription
factor Nanog 5 ECAT5 ES cell expressed Ras (ERas) 6 ECAT7 DNA
(cytosine-5-)-methyltransferase 3-like (Dnmt3l), valiant 1 7 ECAT8
ES cell associated transcript 8 (ECAT8) 8 ECAT9 growth
differentiation factor 3 (Gdf3) 9 ECAT10 SRY-box containing gene 15
(Sox15) 10 ECAT15-1 developmental pluripotency associated 4
(Dppa4), variant 1 11 ECAT15-2 developmental pluripotency
associated 2 (Dppa2) 12 Fthl17 ferritin, heavy polypeptide-like 17
(Fthl17) 13 Sall4 sal-like 4 (Drosophila) (Sall4), transcript
variant a 14 Oct3/4 POU domain, class 5, transcription factor 1
(Pou5f1) 15 Sox2 SRY-box containing gene 2 (Sox2) 16 Rex1 zinc
finger protein 42 (Zfp42) 17 Utf1 undifferentiated embryonic cell
transcription factor 1 (Utf1) 18 Tcl1 T-cell lymphoma breakpoint 1
(Tcl1) 19 Stella developmental pluripotency-associated 3 (Dppa3) 20
Klf4 Kruppel-like factor 4 (gut) (Klf4) 21 .beta.-catenin catenin
(cadherin associated protein), beta 1, 88 kDa (Ctnnb1) 22 c-Myc
myelocytomatosis oncogene (Myc) 23 Stat3 signal transducer and
activator of transcription 3 (Stat3), transcript variant 1 24 Grb2
growth factor receptor bound protein 2 (Grb2)
TABLE-US-00005 TABLE 5 NCBI accession number Name of Number Gene
Characteristic Feature Mouse Human 1 ECAT1 Gene specifically
expressed in ES AB211060 AB211062 cell 2 ECAT2 Gene specifically
expressed in ES NM_025274 NM_001025290 cell 3 ECAT3 Gene
specifically expressed in ES NM_015798 NM_152676 cell 4 ECAT4
Transcription factor having AB093574 NM_024865 homeodomain,
essential factor for differentiation pluripotency maintenance 5
ECAT5 Ras family protein, ES cell growth NM_181548 NM_181532
promoting factor 6 ECAT7 DNA methylation enzyme-related NM_019448
NM_013369 factor, essential for imprinting 7 ECAT8 Gene
specifically expressed in ES AB211061 AB211063 cell, having Tudor
domain 8 ECAT9 Gene specifically expressed in ES NM_008108
NM_020634 cell, belonging to TGF.beta. family 9 ECAT10 Gene
specifically expressed in ES NM_009235 NM_006942 cell, SRY family
transcription factor 10 ECAT15-1 Gene specifically expressed in ES
NM_028610 NM_018189 cell 11 ECAT15-2 Gene specifically expressed in
ES NM_028615 NM_138815 cell 12 Fthl17 Gene specifically expressed
in ES NM_031261 NM_031894 cell, similar to ferritin heavy chain 13
Sall4 Gene specifically expressed in ES NM_175303 NM_020436 cell,
Zn finger protein 14 Oct3/4 POU family transcription factor,
NM_013633 NM_002701 essential for pluripotency maintenance 15 Sox2
SRY family transcription factor, NM_011443 NM_003106 essential for
pluripotency maintenance 16 Rex1 Gene specifically expressed in ES
NM_009556 NM_174900 cell, Zn finger protein 17 Utf1 Transcription
regulation factor NM_009482 NM_003577 highly expressed in ES cell,
promoting growth of ES 18 Tcl1 Oncogene activating AKT, NM_009337
NM_021966 abundantly expressed in ES cell 19 Stella Gene
specifically expressed in ES NM_139218 NM_199286 cell 20 Klf4
Abundantly expressed in ES cell, NM_010637 NM_004235 both actions
as antioncogene and oncogene are reported 21 .beta.-catenin
Transcription factor activated by NM_007614 NM_001904 Wnt signal,
involvement in pluripotency maintenance is reported 22 c-Myc
Transcription control factor NM_010849 NM_002467 participating in
cell proliferation and differentiation and oncogene, involvement in
pluripotency maintenance is reported 23 Stat3 Transcription factor
activated by LIF NM_213659 NM_139276 signal, considered essential
for pluripotency maintenance of mouse ES cells 24 Grb2 Adapter
protein mediating growth NM_008163 NM_002086 factor receptors and
Ras/MAPK cascade
[0145] cDNAs of these genes were inserted into the retroviral
vector pMX-gw by the Gateway technology. First, each of the 24
genes was infected into Fbx15.sup..beta.geo/.beta.geo MEFs, and
then G418 selection was performed under ES cell culture conditions.
However, no G418-resistant colony was obtained. Next, the
retroviral vectors of all of the 24 genes were simultaneously
infected into Fbx15.sup..beta.geo/.beta.geo MEFs. When G418
selection was performed under ES cell culture conditions, a
plurality of drug resistant colonies were obtained. These colonies
were isolated, and cultivation was continued. It was found that
cultivation of these cells over a long period of time could be
performed, and that these cells had morphology similar to that of
ES cells (FIG. 2). In the figure, iPS cells represent induced
pluripotent stem cells (also called "ES like cells", "ES-like
cells", or "ESL cells"), ES represents embryonic stem cells, and
MEF represents differentiated cells (embryonic fibroblasts).
[0146] When expression profiles of the marker genes were examined
by RT-PCR, undifferentiation markers such as Nanog and Oct3/4 were
found to be expressed (FIG. 3). It was found that the expression of
Nanog was close to that of ES cells, whereas the expression of
Oct3/4 was lower than that of ES cells. When DNA methylation status
was examined by the bisulfite genomic sequencing, it was found that
the Nanog gene and Fbx15 gene were highly methylated in MEFs,
whereas they were demethylated in the iPS cells (FIG. 4). About 50%
of IGF2 gene, an imprinting gene, was methylated both in the MEF
and iPS cells. Since it was known that the imprinting memory was
deleted and the IGF2 gene was almost completely demethylated in the
primordial germ cells at 13.5 days after fertilization, from which
the Fbx15.sup..beta.geo/.beta.geo MEFs were isolated, it was
concluded that iPS cells were not derived from primordial germ
cells contaminated in the Fbx15.sup..beta.geo/.beta.geo MEFs. The
above results demonstrated that reprogramming of the differentiated
cells (MEFs) into a state close to that of ES cells was able to be
induced with the combination of the 24 kinds of factors.
[0147] Then, studies were made as to whether or not all of the 24
kinds of genes were required for the reprogramming. With withdrawal
of each individual gene, 23 genes were transfected into the
Fbx15.sup..beta.geo/.beta.geo MEFs. As a result, for 10 genes,
colony formation was found to be inhibited with each withdrawal
thereof (FIG. 5, the gene numbers correspond to the gene numbers
shown in TABLE 4, and the genes are the following 10 kinds of
genes: #3, #4, #5, #11, #14, #15, #18, #20, #21, and #22). When
these ten genes were simultaneously transfected into the
Fbx15.sup..beta.geo/.beta.geo MEFs, G418-resistant colonies were
significantly more efficiently obtained as compared to simultaneous
transfection with the 24 genes.
[0148] Furthermore, 9 genes, withdrawal of each individual gene
from the 10 genes, were transfected into
Fbx15.sup..beta.geo/.beta.geo MEFs. As a result, it was found that
G418-resistant iPS cell colonies were not formed when each of 4
kinds of genes (#14, #15, #20, or #22) was withdrawn (FIG. 6).
Therefore, it was suggested that these four kinds of genes, among
the ten genes, had particularly important roles in the induction of
reprogramming.
Example 2
Induction of Reprogramming with a Combination of 4 Kinds of
Genes
[0149] It was examined whether or not induction of reprogramming of
somatic cells was achievable with the four kinds of genes of which
particular importance was suggested among the 10 genes. By using
the combination of the aforementioned 10 kinds of genes, the
combination of the aforementioned 4 kinds of genes, combinations of
only 3 kinds of genes among the 4 kinds of genes, and combinations
of only 2 kinds of genes among the 4 kinds of genes, these sets of
genes were retrovirally transduced into the MEF cells as somatic
cells in which .beta.geo was knocked into the Fbx15 gene. As a
result, when the 4 kinds of genes were transduced, 160
G418-resistant colonies were obtained. Although this result was
almost the same as that obtained by the transduction with the 10
kinds of genes (179 colonies), the colonies obtained by the 4-gene
transduction were smaller than those by the 10-gene transduction.
When these colonies were passaged, the numbers of colonies having
iPS cell morphology was 9 clones among 12 clones in the case of the
10-gene transduction, whereas there was a somewhat lower tendency
of 7 clones among 12 clones in the case of the 4-gene transduction.
As for the 4 genes, almost the same numbers of iPS cells were
obtained with either of those derived from mouse or those derived
from human.
[0150] When 3 genes selected from the aforementioned 4 genes were
transduced, 36 flat colonies were obtained with one combination
(#14 (Oct3/4), #15 (Sox2), and #20 (Klf4)). However, iPS cells were
not observed when they were passaged. With another combination (#14
(Oct3/4), #20 (Klf4), and #22 (c-Myc)), 54 small colonies were
obtained. When 6 of the relatively large colonies from among those
colonies were passaged, cells similar to ES cells were obtained for
all these 6 clones. However, it seemed that adhesion of the cells
between themselves and to the culture dish was weaker than that of
ES cells. The proliferation rate of the cells was also slower than
that observed in the case of the transduction with the 4 genes.
Further, one colony each was formed with each of the other two
kinds of combinations of 3 genes among the 4 genes. However,
proliferation of the cells was not observed when the cells were
passaged. With any of combinations of 2 genes selected from the 4
genes (6 combinations), no G418-resistant colonies were formed. The
above results are shown in FIG. 7.
[0151] Further, the results of observation of expression profiles
of the ES cell marker genes by RT-PCR are shown in FIG. 10. The
details of the method are as follows. From iPS cells established by
transducing 3 genes (Oct3/4, Klf4, and c-Myc: represented as "Sox2
minus"), 4 genes (Sox2 was added to the three genes: represented as
"4ECAT"), and 10 genes (#3, #4, #5, #11, #18, and #21 in TABLE 4
were added to the four genes: represented as "10ECAT") into
Fbx15.sup..beta.geo/.beta.geo MEFs; iPS cells established by
transducing 10 genes into fibroblasts established from tail tip of
an adult mouse in which .beta.geo was knocked into the Fbx15 gene
(represented as "10ECAT Skin fibroblast"), mouse ES cells, and MEF
cells with no gene transduction, total RNAs were purified, and
treated with DNaseI to remove contamination of genomic DNA. First
strand cDNAs were prepared by a reverse transcription reaction, and
expression profiles of the ES cell marker genes were examined by
PCR. For Oct3/4, Nanog, and ERas, PCR was performed by using
primers which only amplified a transcript product from an
endogenous gene, not from the transduced retrovirus. The primer
sequences are shown in TABLE 6.
TABLE-US-00006 TABLE 6 ECATI ECAT1-RT-S TGT GGG GCC CTG AAA GGC GAG
CTG AGA T (SEQ ID NO: 1) ECAT1-RT-AS ATG GGC CGC CAT ACG ACG ACG
CTC AAC T (SEQ ID NO: 2) Esg1 pH34-U38 GAA GTC TGG TTC CTT GGC AGG
ATG (SEQ ID NO: 3) pH34-L394 ACT CGA TAC ACT GGC CTA GC (SEQ ID NO:
4) Nanog 6047-S1 CAG GTG TTT GAG GGT AGC TC (SEQ ID NO: 5) 6047-AS1
CGG TTC ATC ATG GTA CAG TC (SEQ ID NO: 6) ERas 45328-S118 ACT GCC
CCT CAT CAG ACT GCT ACT (SEQ ID NO: 7) ERas-AS304 CAC TGC CTT GTA
CTC GGG TAG CTG (SEQ ID NO: 8) Gdf3 Gdf3-U253 GTT CCA ACC TGT GCC
TCG CGT CTT (SEQ ID NO: 9) GDF3 L16914 AGC GAG GCA TGG AGA GAG CGG
AGC AG (SEQ ID NO: 10) Fgf4 Fgf4-RT-S CGT GGT GAG CAT CTT CGG AGT
GG (SEQ ID NO: 11) Fgf4-RT-AS CCT TCT TGG TCC GCC CGT TCT TA (SEQ
ID NO: 12) Cripto Cripto-S ATG GAC GCA ACT GTG AAC ATG ATG TTC GCA
(SEQ ID NO: 13) Cripto-AS CTT TGA GGT CCT GGT CCA TCA CGT GAC CAT
(SEQ ID NO: 14) Zfp296 Zfp296-S67 CCA TTA GGG GCC ATC ATC GCT TTC
(SEQ ID NO: 15) Zfp296-AS350 CAC TGC TCA CTG GAG GGG GCT TGC (SEQ
ID NO: 16) Dax1 Dax1-S1096 TGC TGC GGT CCA GGC CAT CAA GAG (SEQ ID
NO: 17) Dax1-AS1305 GGG CAC TGT TCA GTT CAG CGG ATC (SEQ ID NO: 18)
Oct3/4 Oct3/4-S9 TCT TTC CAC CAG GCC CCC GGC TC (SEQ ID NO: 19)
Oct3/4-AS210 TGC GGG CGG ACA TGG GGA GAT CC (SEQ ID NO: 20) NAT1
NAT1 U283 ATT CTT CGT TGT CAA GCC GCC AAA GTG GAG (SEQ ID NO: 21)
NAT1 L476 AGT TGT TTG CTG CGG AGT TGT CAT CTC GTC (SEQ ID NO:
22)
[0152] The results shown in this figure revealed that, by
transduction of the 3 genes, expression of each of ERas and Fgf4
was efficiently induced, but expression of each of Oct3/4 and
Nanog, essential factors for the maintenance of pluripotency, was
not induced, or was very weak even when induced. However, when the
4 genes were transduced, there was one clone (#7) in which Oct3/4
and Nanog were relatively strongly induced among 4 clones examined.
Further, when the 10 genes were transduced, strong induction of
each of Oct3/4 and Nanog was observed in 3 clones among 5 clones
examined.
[0153] These results revealed that a combination of at least 3
genes (#14 (Oct3/4), #20 (Klf4), and #22 (c-Myc)) was essential for
reprogramming under these conditions, and in the cases of the
4-gene group and 10-gene group including the 3 kinds of genes, the
reprogramming efficiency was increased in proportion to the
increasing number of genes. In other words, in accordance with the
guidance disclosed herein, the minimum combination of nuclear
reprogramming factors required for iPS cell induction under a given
set of experimental conditions could be further optimized as
evidenced below.
Example 3
Analysis of Pluripotency of Reprogrammed Cells
[0154] In order to evaluate the differentiation pluripotency of the
established iPS cells, the iPS cells established with 24 factors,
10 factors, and 4 factors were subcutaneously transplanted into
nude mice. As a result, tumors having a size similar to that
observed with ES cells were formed in all animals. Histologically,
the tumors consisted of a plurality of kinds of cells, and
cartilaginous tissues, nervous tissues, muscular tissues, fat
tissues, and intestinal tract-like tissues were observed (FIG. 8),
which verified pluripotency of the iPS cells. In contrast, although
tumors were formed when the cells established with the 3 factors
were transplanted into nude mice, they were formed histologically
only from undifferentiated cells. These results suggested that a
Sox family gene was essential for the induction of differentiation
pluripotency.
Example 4
Reprogramming of Fibroblasts Derived from Tails of Adult Mice
[0155] The 4 factors identified in the mouse embryonic fibroblasts
(MEFs) were transduced into fibroblasts derived from tails of
.beta.geo knockin Fbx15 adult mice systemically expressing green
fluorescence protein (GFP). Then, the cells were cultured on feeder
cells under the same conditions as ES cell culture conditions, and
G418 selection was performed. In about two weeks after the start of
the drug selection, a plurality of colonies of iPS cells were
obtained. When these cells were subcutaneously transplanted to nude
mice, teratomas consisting of a variety of all three germ layer
tissues were formed. Further, when the iPS cells derived from adult
dermal fibroblasts were transplanted to the blastocysts, and then
transplanted into the uteri of pseudopregnant mice, embryos in
which the GFP-positive cells were systemically distributed were
observed among those at 13.5 days after fertilization (FIG. 9),
demonstrating that the iPS cells had pluripotency and were able to
contribute to mouse embryogenesis. These results indicate that the
identified class of factors had an ability to induce reprogramming
of not only somatic cells in an embryonic period, but also somatic
cells of mature mice. Practically, it is extremely important that
the reprogramming can be induced in cells derived from adult
skin.
Example 5
Effect of Cytokine on iPS Cell Establishment
[0156] An effect of cytokine on iPS cell establishment was
investigated. Expression vector (pMX retroviral vector) for basic
fibroblast growth factor (bFGF) or stem cell factor (SCF) was
transduced into feeder cells (STO cells) to establish cells
permanently expressing the cytokines. MEFs derived from the
Fbx15.sup..beta.geo/.beta.geo mouse (500,000 cells/100 mm dish)
were cultured on these STO cells and transduced with the 4 factors,
and then subjected to G418 selection. As a result, the number of
formed colonies increased 20 times or higher on the STO cells
producing bFGF (FIG. 11) or SCF (data not shown), as compared with
the culture on normal STO cells. Further, although no iPS cell
colony was formed on the normal STO cells when the 3 factors other
than c-Myc were transduced, colony formation was observed on the
STO cells producing bFGF (FIG. 11) or SCF (data not shown). These
results revealed that stimulation with the cytokine increased the
efficiency of the establishment of iPS cells from MEFs, and the
nuclear reprogramming was achievable by using a cytokine instead of
c-Myc.
Example 6
IFS Cell Generation with Other Oct, Klf, Myc, and Sox Family
Members
[0157] Family genes exist for all of the Oct3/4, Klf4, c-Myc, and
Sox2 genes (TABLES 1 and 2). Accordingly, studies were made as to
whether iPS cells could be established with the family genes
instead of the 4 genes. In TABLE 7, combined experimental results
in duplicate are shown. With regard to the Sox family, Sox1 gave
almost the same number of G418-resistant colonies formed and iPS
cell establishment efficiency as those with Sox2. As for Sox3, the
number of G418-resistant colonies formed was about 1/10 of that
with Sox2, however, iPS cell establishment efficiency of the
colonies picked up was in fact higher than that with Sox2. As for
Sox15, both the number of G418-resistant colonies formed and iPS
cell establishment efficiency were lower than those with Sox2. As
for Sox17, the number of G418-resistant colonies formed was almost
the same as that with Sox2, however, iPS cell establishment
efficiency was low. With regard to the Klf family, Klf2 gave a
smaller number or G418-resistant colonies than Klf4, however, they
gave almost the same iPS cell establishment efficiency. With regard
to the Myc family, it was found that wild-type c-Myc was almost the
same as a T58A mutant both in the number of G418-resistant colonies
formed and iPS cell establishment efficiency. Further, each of
N-Myc and L-Myc (each wild type) was almost the same as c-Myc in
both of the number of G418-resistant colonies formed and iPS cell
establishment efficiency.
TABLE-US-00007 TABLE 7 Number Number Number of iPS cell Transduced
of formed of picked established iPS establishment gene colonies
colonies cell strain efficiency (%) 4 Factors 85 12 5 42 (cMycT58A)
Sox1 84 12 7 58 Sox3 8 8 7 92 Sox15 11 11 1 8 Sox17 78 12 2 17 Klf2
11 10 5 50 c-MycWT 53 11 8 72 N-MycWT 40 12 7 58 L-MycWT 50 12 11
92 3 Factors 6 6 2 17 (-Sox2)
Example 7
Use of a Nanog-GFP-Puro.sup.r Reporter to Establish iPS Cells
[0158] Studies were made as to whether iPS cells could be
established with a reporter other than Fbx15-.beta.geo.
Escherichia. coli artificial chromosome (BAC) containing the Nanog
gene in the center was isolated, and then the GFP gene and the
puromycin resistance gene were knocked in by recombination in E.
coli (FIG. 12A). Subsequently, the above modified BAC was
introduced into ES cells to confirm that the cells became
GFP-positive in an undifferentiated state specific manner (data not
shown). Then, these ES cells were transplanted in mouse blastocysts
to create transgenic mice via chimeric mice. In these mice,
GFP-positive cells were specifically observed in inner cell masses
of the blastocysts or gonads of embryos at 13.5 days after
fertilization (FIG. 12B). The gonads were removed from the embryos
at 13.5 days after fertilization (hybrid of DBA, 129, and C57BL/6
mice), and MEFs were isolated. The isolated MEFs were confirmed to
be GFP-negative (FIG. 13) by flow cytometry. These MEFs were
retrovirally transduced with the 4 factors and subjected to
puromycin selection, and as a result, a plural number of resistant
colonies were obtained. Only about 10 to 20% of the colonies were
GFP-positive. When the GFP-positive colonies were passaged, they
gave morphology (FIG. 14) and proliferation (FIG. 15) similar to
those of ES cells. Examination of the gene expression pattern
revealed that the expression pattern was closer to that of ES cells
as compared to the iPS cells isolated from
Fbx15.sup..beta.geo/.beta.geo MEFs by G418 selection (FIG. 16).
When these cells were transplanted to nude mice, teratoma formation
was induced, thereby the cells were confirmed to be iPS cells (FIG.
17). Further, chimeric mice were born by transplanting the iPS
cells obtained by Nanog-GFP selection to the blastocysts of C57BL/6
mice (FIG. 18). When these chimeric mice were mated, germ-line
transmission was observed (FIG. 19). In these iPS cells established
by Nanog-GFP selection, which were closer to ES cells, the
expressions of the 4 factors from the retroviruses were almost
completely silenced, suggesting that self-replication was
maintained by endogenous Oct3/4 and Sox2.
Example 8
In Vitro Differentiation Induction
[0159] Confluent iPS cells in 10 cm dishes were trypsinized and
suspended in ES cell medium (the STO cells were removed by adhesion
to a gelatin-coated dish for 10 to 20 minutes after the
suspension). 2.times.10.sup.6 cells were cultured for four days in
a HEMA (2-hydroxyethyl methacrylate) coated E. coli culture dish as
a suspension culture to form embryoid bodies (EBs) (day 1 to 4). On
the 4th day of EB formation (day 4), all of the EBs were
transferred to a 10-cm tissue culture dish, and cultured in ES cell
medium for 24 hours to allow adhesion. After 24 hours (day 5), the
medium was changed to an ITS/fibronectin-containing medium. The
culture was performed for 7 days (medium was exchanged every 2
days), and nestin-positive cells were selected (cells of other
pedigrees were dying to some extent in a culture under serum-free
condition) (day 5 to 12). A2B5-positive cells were then induced.
After 7 days (day 12), the cells were separated by trypsinization,
and the remaining EBs were removed. 1.times.10.sup.5 cells were
seeded on a poly-L-ornithine/fibronectin-coated 24-well plate, and
cultured for 4 days in an N2/bFGF-containing medium (medium was
exchanged every 2 days) (day 12 to 16). After 4 days (day 16), the
medium was changed to an N2/bFGF/EGF-containing medium, and the
culture was continued for 4 days (medium was exchanged every 2
days) (day 16 to 20). After 4 days (day 20), the medium was changed
to an N2/bFGF/PDGF-containing medium, and the culture was continued
for 4 days (medium was exchanged every 2 days) (day 20 to 24).
During this period (day 12 to 24), when the cells had increased
excessively and reached confluent, they were passaged at
appropriate times, and 1 to 2.times.10.sup.5 cells were seeded (the
number of the cells varied depending on the timing of the passage).
After 4 days (day 24), the medium was changed to an N2T3 medium,
and the culture was continued for 7 days (day 24 to 31) with medium
exchange every 2 days. On day 31, the cells were fixed and
subjected to immunostaining. As a result, differentiation of the
iPS cells into .beta.III tubulin-positive nerve cells, O4-positive
oligodendrocytes, and GFAP-positive astrocytes was observed (FIG.
20).
Example 9
Establishment of iPS Cells without Drug Selection
[0160] In order to establish iPS cells from arbitrary mouse somatic
cells other than those derived from the Fbx15-.beta.geo knockin
mouse, a method for the establishment without using drug selection
was developed. 10,000, 50,000, or 100,000 cells mouse embryo
fibroblasts (MEFs) were cultured on a 10 cm dish (on STO feeder
cells). This is less than the number of cells used above, Control.
DNA or the 4 factors were retrovirally transduced. When culture was
performed for 2 weeks in the ES cell medium (without G418
selection), no colony formation was observed in the dish in which
the control DNA was transduced, whilst in the dish in which the 4
factors were transduced, a plurality of compact colonies were
formed as well as flat colonies considered to be transformed (FIG.
21). When 24 colonies were picked up from these colonies and
culture was continued, ES cell-like morphology was observed. Gene
expression profiles thereof were examined by RT-PCR, and as a
result, the expression of Esg1, an ES cell marker, was observed in
7 clones. Induction of many ES cell markers such as Nanog, ERas,
GDF3, Oct3/4, and Sox2 was observed in clone 4, and therefore the
cells were considered to be iPS cells (FIG. 22). The above results
demonstrated that drug selection using Fbx15-.beta.geo knockin or
the like was not indispensable for iPS cell establishment, and iPS
cells could be established from arbitrary mouse-derived somatic
cells. This also suggested the possibility that iPS cells could be
established from somatic cells of a disease model mouse by the
aforementioned technique.
Example 10
iPS Cell Generation from Hepatocytes and Gastric Mucous Cells
[0161] As cells from which iPS cells were induced, hepatocytes and
gastric mucous cells being cells other than fibroblasts were
examined. Hepatocytes were isolated from the liver of the
Fbx15.sup..beta.geo/.beta.geo mice by perfusion. These hepatocytes
were retrovirally introduced with the 4 factors, and then subjected
to G418 selection to obtain plural iPS cell colonies. As a result
of gene expression pattern analysis using a DNA microarray, the iPS
cells derived from the liver were found to be more similar to ES
cells than the iPS cells derived from dermal fibroblasts or
embryonic fibroblasts. iPS cells were obtained also from gastric
mucous cells in the same manner as those from hepatocytes.
Example 11
Effect of MAP Kinase Inhibitor on iPS Cell Establishment
[0162] PD98059 is an inhibitor of MAP kinase which suppresses
proliferation of various differentiated cells. However, it is known
to promote maintenance of undifferentiated status and proliferation
of ES cells. Effects of PD98059 on iPS cell establishment were thus
examined. MEFs established from a mouse having the selective
markers of Nanog-EGFP-IRES-Puro were retrovirally introduced with
the 4 factors and subjected to puromycin selection. When PD98059
was not added, the percentage of GFP-positive colonies was 8% of
the iPS cell colonies obtained. However, in the group to which
PD98059 (final concentration: 25 .mu.M) was continuously added from
the next day of the retroviral transfection, 45% of the colonies
obtained were GFP-positive. The results were interpreted to be due
to PD98059 promoting the proliferation of the GFP-positive iPS
cells, which are closer to ES cells, whilst PD98059 suppressing the
proliferation of the GFP-negative iPS cells or differentiated
cells. From these results, PD98059 was demonstrated to be able to
be used for establishment of the iPS cells closer to ES cells or
establishment of iPS cells without using drug selection.
Example 12
Establishment of iPS Cells from Embryonic HDFs in Mouse ES Cell
Medium
[0163] A plasmid, containing the red fluorescence protein gene
downstream from the mouse Oct3/4 gene promoter and the hygromycin
resistance gene downstream from the PGK promoter, was introduced by
nucleofection into embryonic human dermal fibroblasts (HDFs) in
which solute carrier family 7 (Slc7a1, NCBI accession number
NM.sub.--007513) as a mouse ecotropic virus receptor was expressed
by lentiviral transduction. Hygromycin selection was performed to
establish strains with stable expression. 800,000 cells were seeded
on the STO cells treated with mitomycin, and on the next day,
Oct3/4, Sox2, Klf4, and c-Myc (each derived from human) were
retrovirally transduced into the cells. 24 colonies were picked up
from those obtained after 3 weeks (FIG. 23, left), and transferred
on a 24-well plate on which the STO cells were seeded and then
cultured. After 2 weeks, one grown clone was passaged on a 6-well
plate on which the STO cells were seeded and cultured. As a result,
cells morphologically similar to ES cells were obtained (FIG. 23,
right), suggesting that the cells were iPS cells. The mouse ES cell
medium was used as every medium.
Example 13
Establishment of iPS Cells from Adult HDFs in Mouse ES Cell
Medium
[0164] Human adult dermal fibroblasts (adult HDFs) or human
neonatal foreskin cells (BJ) were transduced with Slc7a1 (mouse
retroviral receptor) by using lentivirus, and the resulting cells
were seeded on 800,000 feeder cells (mitomycin-treated STO cells).
The genes were retrovirally transduced as the following
combinations.
[0165] 1. Oct3/4, Sox2, Klf4, c-Myc, TERT, and SV40 Large T
antigen
[0166] 2. Oct3/4, Sox2, Klf4, c-Myc, TERT, HPV16 E6
[0167] 3. Oct3/4, Sox2, Klf4, c-Myc, TERT, HPV16 E7
[0168] 4. Oct3/4, Sox2, Klf4, c-Myc, TERT, HPV16 E6, HPV16 E7
[0169] 5. Oct3/4, Sox2, Klf4, c-Myc, TERT, Bmi1
[0170] (Oct3/4, Sox2, Klf4, c-Myc and TERT were derived from human,
and Bmi1 was derived from mouse)
[0171] The culture was continued under the culture conditions for
mouse ES cells without drug selection. As a result, colonies
considered to be those of iPS cells emerged on the 8th day after
the virus transfection on the dish in which the factors were
introduced according to Combination 1 (FIG. 24). iPS cell-like
colonies also emerged with the other combinations (2 to 5),
although they were not as apparent when compared to Combination 1.
When only the 4 factors were transduced, no colonies emerged. Cells
transduced with only the four factors under the experimental
conditions used in this Example showed only faint staining for
alkaline phosphatase (FIG. 25(A)-(B)).
[0172] However, optimization of the methods heretofore described
revealed successful induction of iPS cells through staining of any
number of pluripotent markers, including alkaline phosphatase,
ABCG-2, E-cadherin, SSEA-3, and SSEA-4 when adult human dermal
fibroblasts expressing mouse Slc7a1 gene were generated into iPS
cells by reprogramming with the four factors plus TERT and SV40
Large T antigen (i.e. six factors total: c-Myc, Klf4, Sox2, Oct3/4,
TERT, and SV40 Large T antigen) (FIG. 26(A)-(B)). These cells were
assessed for pluripotent cell markers (FIG. 27). These cells were
found to express ECATS, including Nanog and ESG1. Similarly, BJ
fibroblasts expressing mouse Slc7a1 gene were generated into iPS
cells by reprogramming with the following same factors. These cells
were also tested for pluripotent cell markers (FIG. 28). In
addition, iPS cells generated from adult HDFs were selected for
subcutaneous injection into the dorsal flanks of SCID mice.
Teratoma formation was observed (FIGS. 29(A)-(D)). Human dermal
fibroblasts were also shown to differentiate in vitro upon
culturing in HEMA-coated plates (7 days) and gelatinized dishes (7
days) (FIG. 30).
Example 14
Optimization of Retroviral Transduction for Generating Human iPS
Cells
[0173] Next, iPS cell generation from adult human somatic cells was
further evaluated by optimizing retroviral transduction in human
fibroblasts and subsequent culture conditions. Induction of iPS
cells from mouse fibroblasts requires retroviruses with high
transduction efficiencies (Takahashi et al., Cell 126: 663-676,
2006). Therefore, transduction methods in adult human dermal
fibroblasts (HDFs) were optimized. First, green fluorescent protein
(GFP) was introduced into adult HDF with amphotropic retrovirus
produced in PLAT-A packaging cells. As a control, GFP was
introduced into mouse embryonic fibroblasts (MEF) with ecotropic
retrovirus produced in PLAT-E packaging cells (Morita et al., Gene
Ther. 7:1063-66, 2000). In MEF, more than 80% of cells expressed
GFP (FIG. 31). In contrast, less than 20% of HDF expressed GFP with
significantly lower intensity than in MEF. To improve the
transduction efficiency, the mouse receptor for retroviruses,
Slc7a1 (Verrey et al., Pflugers Arch. 447:532-542, 2004) (also
known as mCAT1), was introduced into HDF with lentivirus. Then GFP
was introduced into HDF-Slc7a1 with ecotropic retrovirus. This
strategy yielded a transduction efficiency of 60%, with a similar
intensity to that in MEF.
Example 15
Generation of iPS Cells from Adult HDFs in Primate ES Cell Culture
Medium
[0174] The protocol for human iPS cell induction is summarized in
FIG. 32A. pMXs encoding human Oct3/4, Sox2, Klf4, and c-Myc were
introduced into HDF-Slc7a1 cells (FIG. 32B; 8.times.10.sup.5 cells
per 100 mm dish). The HDFs were derived from facial dermis of
36-year-old Caucasian female.
[0175] Six days after transduction, the cells were harvested by
trypsinization and plated onto mitomycin C-treated SNL feeder cells
(McMahon et al., Cell 62:1073-85, 1990) at 5.times.10.sup.4 or
5.times.10.sup.5 cells per 100 mm dish. The next day, the medium
(DMEM containing 10% FBS) was replaced with a medium for primate ES
cell culture supplemented with 4 ng/ml basic fibroblast growth
factor (bFGF). Approximately two weeks later, some granulated
colonies appeared that were not similar to hES cells in morphology
(FIG. 32C). Around day 25, distinct types of colonies that were
flat and resembled hES cell colonies were observed (FIG. 32D). From
5.times.10.sup.4 fibroblasts, .about.10 hES cell-like colonies and
.about.100 granulated colonies (7/122, 8/84, 8/171, 5/73, 6/122,
and 11/213 in six independent experiments, summarized in TABLE 8)
were observed.
TABLE-US-00008 TABLE 8 Summary of the iPS cell induction
experiments Cell No. No. of No. of No. of No. of Exp. Parental
seeded ES-like total picked up established ID cells at d 6 colony
colony colony clone 201B HDF 50000 7 129 7 5 243H HFLS 500000 0
>1000 50000 17 679 6 2 246B HDF 500000 0 420 500000 2 508 50000
8 92 6 6 246G BJ 50000 7 10 6 5 500000 86 98 500000 106 108 249D
HDF 500000 0 320 500000 0 467 50000 8 179 6 4 253F HDF 50000 5 78 3
2 50000 6 128 3 3 500000 10 531 500000 3 738 282C HDF 50000 11 224
3 1 282H BJ 50000 13 15 3 2 282R HFLS 5000 31 98 6 2
[0176] At day 30, hES cell-like colonies were picked up and
mechanically disaggregated into small clumps without enzymatic
digestion. When starting with 5.times.10.sup.5 fibroblasts, the
dish was nearly covered with more than 300 granulated colonies.
Occasionally some hES cell-like colonies in between the granulated
cells were observed, but it was difficult to isolate hES cell-like
colonies because of the high density of granulated cells.
[0177] The hES-like cells expanded on SNL feeder cells under the
human ES cell culture condition. They formed tightly packed and
flat colonies (FIG. 32E). Each cell exhibited morphology similar to
that of human ES cells, characterized by large nucleoli and scant
cytoplasm (FIG. 32F). As is the case with hES cells, occasionally
spontaneous differentiation was observed in the center of the
colony (FIG. 32G).
[0178] These cells also showed similarity to hES cells in feeder
dependency. They did not attach to gelatin-coated tissue-culture
plates. By contrast, they maintained an undifferentiated state on
Matrigel-coated plates in MEF-conditioned medium (MEF-CM), but not
in ES medium (FIG. 33).
[0179] Since these cells were indistinguishable from hES cells in
morphology and other aspects noted above, the selected cells after
transduction of HDFs are referred to as human iPS cells. The
molecular and functional evidence for this claim is further
described below. Human iPS cells clones established in this study
are summarized in TABLE 9.
TABLE-US-00009 TABLE 9 Characterization of established clones
Marker expression Pluripotency RT- Cardio- Tera- Clone Source PCR
IC EB PA6 myocyte toma 201B1 HDF 201B2 201B3 201B6 201B7 243H1 HFLS
243H7 246B1 HDF 246B2 246B3 246B4 246B5 246B6 246G1 BJ 246G3 246G4
246G5 246G6 253F1 HDF 253F2 253F3 253F4 253F5 IC;
immunocytochemistry, EB; embryoid body
[0180] Human iPS Cells Express hES Markers
[0181] In general, except for a few cells at the edge of the
colonies, human iPS cells did not express stage-specific embryonic
antigen (SSEA)-1 (FIG. 32H). In contrast, they expressed hES
cell-specific surface antigens (Adewumi et al., Nat. Biotechnol.
25:803-816, 2007), including SSEA-3, SSEA-4, tumor-related antigen
(TRA)-1-60, TRA-1-81, and TRA-2-49/6E (alkaline phosphatase), and
NANOG protein (FIG. 32I-N).
[0182] RT-PCR showed human iPS cells expressed many
undifferentiated ES cell gene markers (Adewumi et al., Nat.
Biotechnol. 25:803-816, 2007), such as OCT3/4, SOX2, NANOG, growth
and differentiation factor 3 (GDF3), reduced expression 1 (REX1),
fibroblast growth factor 4 (FGF4), embryonic cell-specific gene 1
(ESG1), developmental pluripotency-associated 2 (DPPA2), DPPA4, and
telomerase reverse transcriptase (hTERT) at levels equivalent to or
higher than those in the human embryonic carcinoma cell line,
NTERA-2 (FIG. 34A). By western blotting, proteins levels of OCT3/4,
SOX2, NANOG, SALL4, E-CADHERIN, and hTERT were similar in human iPS
cells and hES cells (FIG. 34B). In human iPS cells, the expression
of transgenes from integrated retroviruses was efficiently
silenced, indicating that they depend on the endogenous expression
of these genes (FIG. 34C).
[0183] Promoters of ES Cell-Specific Genes are Active in Human iPS
Cells
[0184] Bisulfite genomic sequencing analyses evaluating the
methylation statuses of cytosine guanine dinucleotides (CpG) in the
promoter regions of pluripotent-associated genes, such as OCT3/4,
REX1, and NANOG, revealed that they were highly unmethylated,
whereas the CpG dinucleotides of the regions were highly methylated
in parental HDFs (FIG. 34D). These findings indicate that these
promoters are active in human iPS cells.
[0185] Luciferase reporter assays also showed that human OCT3/4 and
REX1 promoters had high levels of transcriptional activity in human
iPS cells, but not in HDF. The promoter activities of ubiquitously
expressed genes, such as human RNA polymerase II (PolII), showed
similar activities in both human iPS cells and HDF (FIG. 34E).
[0186] High Telomerase Activity and Exponential Growth of Human iPS
Cells
[0187] As predicted from the high expression levels of hTERT, human
iPS cells showed high telomerase activity (FIG. 35A). They
proliferated exponentially for at least 4 months (FIG. 35B). The
calculated doubling time of human iPS cells were 46.9.+-.12.4
(clone 201B2), 47.8.+-.6.6 (201B6) and 43.2.+-.11.5 (201B7) hours
(FIG. 35B). These times are equivalent to the reported doubling
time of hES cells (Cowan et al., N. Engl. J. Med. 350:1353-56,
2004).
[0188] Human iPS Cells Are Derived from HDF, not
Cross-Contamination
[0189] PCR of genomic DNA of human iPS cells showed that all clones
have integration of all the four retroviruses (FIG. 36A). Southern
blot analysis with a c-Myc cDNA probe revealed that each clone had
a unique pattern of retroviral integration sites (FIG. 36B). In
addition, the patterns of 16 short tandem repeats were completely
matched between human iPS clones and parental HDF (TABLE 10).
TABLE-US-00010 TABLE 10 STR analyses of HDF-derived iPS cells Clone
Locus 201B1 201B2 201B3 201B6 201B7 NTERA-2 HDF D3S1358 15 17 15 17
15 17 15 17 15 17 15 15 17 TH01 5 5 5 5 5 9 5 D21S11 28 28 28 28 28
29 30 28 D18S51 14 14 14 14 14 13 14 Penta_E 7 19 7 19 7 19 7 19 7
19 5 14 7 19 D5S818 11 11 11 11 11 8 11 11 D13S317 10 14 10 14 10
14 10 14 10 14 14 10 14 D7S820 9 10 9 10 9 10 9 10 9 10 12 9 10
D16S539 11 13 11 13 11 13 11 13 11 13 11 16 11 13 CSF1PO 10 10 10
10 10 9 11 10 Penta_D 8 10 8 10 8 10 8 10 8 10 11 12 8 10 AMEL X X
X X X X Y X vWA 15 18 15 18 15 18 15 18 15 18 19 15 18 D8S1179 8 10
8 10 8 10 8 10 8 10 13 15 8 10 TPOX 8 9 8 9 8 9 8 9 8 9 8 9 8 9 FGA
20 22 20 22 20 22 20 22 20 22 23 20 22 These patterns differed from
any established hES cell lines reported on National Institutes of
Health website
(http://stemcells.nih.gov/research/nihresearch/scunit/genotyping.htm).
In addition, chromosomal G-band analyses showed that human iPS
cells had a normal karyotype of 46XX (not shown). Thus, human iPS
clones were derived from HDF and were not a result of
cross-contamination.
Example 16
Embryoid Body-Mediated Differentiation of Human iPS Cells
[0190] To determine the differentiation ability of human iPS cells
in vitro, floating cultivation was used to form embryoid bodies
(EBs) (Itskovitz-Eldor et al., Mol. Med. 6:88-95, 2000). After 8
days in suspension culture, iPS cells formed ball-shaped structures
(FIG. 37A). These embryoid body-like structures were transferred to
gelatin-coated plates and continued cultivation for another 8 days.
Attached cells showed various types of morphologies, such as those
resembling neuronal cells, cobblestone-like cells, and epithelial
cells (FIG. 37B-E). Immunocytochemistry detected cells positive for
.beta.III-tubulin (a marker of ectoderm), glial fibrillary acidic
protein (GFAP, ectoderm), .alpha.-smooth muscle actin (.alpha.-SMA,
mesoderm), desmin (mesoderm), .alpha.-fetoprotein (AFP, endoderm),
and vimentin (mesoderm and parietal endoderm) (FIG. 37F-K). RT-PCR
confirmed that these differentiated cells expressed forkhead box A2
(FOXA2, a marker of endoderm), AFP (endoderm), cytokeratin 8 and 18
(endoderm), SRY-box containing gene 17 (SOX17, endoderm), BRACHYURY
(mesoderm), Msh homeobox 1 (MSX/, mesoderm), microtubule-associated
protein 2 (MAP2, ectoderm), and paired box 6 (PAX6, ectoderm) (FIG.
37L). In contrast, expression of OCT3/4, SOX2, and NANOG was
significantly decreased. These data demonstrated that iPS cells
could differentiate into three germ layers in vitro.
Example 17
Directed Differentiation of Human iPS Cells into Neural Cells
[0191] Next, it was examined whether lineage-directed
differentiation of human iPS cells could be induced by reported
methods for hES cells. Human iPS cells were seeded on PA6 feeder
layer and maintained under differentiation conditions for 2 weeks
(Kawasaki et al., Nueron 28:31-40, 2000). Cells spread drastically,
and some neuronal structures were observed (FIG. 38A).
Immunocytochemistry detected cells positive for tyrosine
hydroxylase and .beta.III tubulin in the culture (FIG. 38B). PCR
analysis revealed expression of dopaminergic neuron markers, such
as aromatic-L-amino acid decarboxylase (AADC), choline
acetyltransferase (ChAT), solute carrier family 6 (neurotransmitter
transporter, dopamine), member 3 (DAT), and LIM homeobox
transcription factor 1 beta (LMXIB), as well as another neuron
marker, MAP2 (FIG. 38C). In contrast, GFAP expression was not
induced with this system. On the other hand, expression of OCT3/4,
SOX2, and NANOG decreased (FIG. 38C). These data demonstrated that
iPS cells could differentiate into neuronal cells, including
dopaminergic neurons, by co-culture with PA6 cells.
Example 18
Directed Differentiation of Human iPS Cells into Cardiac Cells
[0192] Next directed cardiac differentiation of human iPS cells was
examined with the recently reported protocol, which utilizes
activin A and bone morphogenetic protein (BMP) 4 (Laflamme et al.,
Nat. Biotechnol. 25:1015-24, 2007). Twelve days after the induction
of differentiation, clumps of cells started beating (FIG. 38D).
RT-PCR showed that these cells expressed cardiomyocyte markers,
such as troponin T type 2 cardiac (TnTc); myocyte enhancer factor
2C (MEF2C); NK2 transcription factor related, locus 5 (NKX2.5)
myosin, light polypeptide 7, regulatory (MYL2A), and myosin, heavy
polypeptide 7, cardiac muscle, beta (MYHCB) (FIG. 38E). In
contrast, the expression of Oct3/4, Sox2, and Nanog markedly
decreased. Thus, human iPS cells can differentiate into cardiac
myocytes in vitro.
Example 19
Teratoma Formation from Human in Cells
[0193] To test pluripotency in vivo, human iPS cells (clone 201B7)
were transplanted subcutaneously into dorsal flanks of
immunodeficient (SCID) mice. Nine weeks after injection, tumor
formation was observed. Histological examination showed that the
tumor contained various tissues (FIG. 39), including gut-like
epithelial tissues (endoderm), striated muscle (mesoderm),
cartilage (muscle), neural tissues (ectoderm), and
keratin-containing squamous tissues (ectoderm).
Example 20
Generation of iPS Cells from Other Human Somatic Cells
[0194] In addition to HDF, primary human fibroblast-like
synoviocytes (HFLS) from synovial tissue of 69-year-old Caucasian
male and BJ cells, a cell line established from neonate
fibroblasts, were used (TABLE 8). From 5.times.10.sup.4 HFLS cells
per 100 mm dish, more than 600 hundred granulated colonies and 17
hES cell-like colonies were obtained. Six colonies were picked, of
which only two were expandable as iPS cells (FIG. 40). Dishes
seeded with 5.times.10.sup.5 HFLS were covered with granulated
cells, and no hES cell-like colonies were distinguishable. In
contrast, 7 to 8 and .about.100 hES cell-like colonies were
obtained from 5.times.10.sup.4 and 5.times.10.sup.5 BJ cells,
respectively, with only a few granulated colonies (TABLE 8). Six
hES cell-like colonies were picked and iPS cells were generated
from five colonies (FIG. 40). Human iPS cells derived from HFLS and
BJ expressed hES cell-marker genes at levels similar to or higher
than those in hES cells (FIG. 41). They differentiated into all
three germ layers through EBs (FIG. 42). STR analyses confirmed
that iPS-HFLS cells and iPS-BJ cells were derived from HFLS and BJ
fibroblasts, respectively (TABLE 11 and TABLE 12).
TABLE-US-00011 TABLE 11 STR analyses of HFLS-derived iPS cells
Locus Clone 243H1 243H7 HFLS D3S1358 16 17 16 17 16 17 TH01 5 9 5 9
5 9 D21S11 28 30 28 30 28 30 D18S51 14 17 14 17 14 17 Penta_E 5 12
5 12 5 12 D5S818 10 12 10 12 10 12 D13S317 13 13 13 D7S820 9 12 9
12 8 12 D16S539 11 13 11 13 11 13 CSF1PO 10 11 10 11 10 11 Penta_D
9 11 9 11 9 11 AMEL X X Y X Y vWA 17 19 17 19 17 19 D8S1179 13 13
13 TPOX 8 11 8 11 8 11 FGA 21 22 21 22 21 22
TABLE-US-00012 TABLE 12 STR analyses of BJ-derived iPS cells Clone
Locus 246G1 246G3 246G4 246G5 246G6 BJ D3S1358 13 15 13 15 13 15 13
15 13 15 13 15 TH01 6 7 6 7 6 7 6 7 6 7 6 7 D21S11 28 28 28 28 28
28 D18S51 16 18 16 18 16 18 16 18 16 18 16 18 Penta_E 7 17 7 17 7
17 7 17 7 17 7 17 D5S818 11 11 11 11 11 11 D13S317 9 10 9 10 9 10 9
10 9 10 9 10 D7S820 11 12 11 12 11 12 11 12 11 12 11 12 D16S539 9
13 9 13 9 13 9 13 9 13 9 13 CSF1PO 9 11 9 11 9 11 9 11 9 11 9 11
Penta_D 11 12 11 12 11 12 11 12 11 12 11 12 AMEL X Y X Y X Y X Y X
Y X Y vWA 16 18 16 18 16 18 16 18 16 18 16 18 D8S1179 9 11 9 11 9
11 9 11 9 11 9 11 TPOX 10 11 10 11 10 11 10 11 10 11 10 11 FGA 22
23 22 23 22 23 22 23 22 23 22 23
[0195] Thus, with Examples 13-20 it was shown that iPS cells can be
generated from adult HDF and other somatic cells by retroviral
transduction of the same four transcription factors, namely Oct3/4,
Sox2, Klf4, and c-Myc. The established human iPS cells are
indistinguishable from hES cells in many aspects, including
morphology, proliferation, feeder dependence, surface markers, gene
expression, promoter activities, telomerase activities, in vitro
differentiation, and teratoma formation. The four retroviruses are
nearly completely silenced in human iPS cells, indicating that
these cells are fully reprogrammed and do not depend on continuous
expression of the transgenes for self-renewal.
[0196] hES cells are different from mouse counterparts in many
respects (Rao, M., Dev. Biol. 275:269-286, 2004). hES cell colonies
are flatter and do not override each other. hES cells depend on
bFGF for self renewal (Amit et al., Dev. Biol. 227:271-78, 2000),
whereas mouse ES cells depend on the LIF/Stat3 pathway (Matsuda et
al., EMBO J. 18:4261-69, 1999; Niwa et al., Genes Dev. 12:2048-60,
1998). BMP induces differentiation in hES cells (Xu et al., Nat.
Methods 2:185-90, 2005) but is involved in self renewal of mouse ES
cells (Ying et al., Cell 115:281-92, 2003). Because of these
differences, it has been speculated that factors required for
reprogramming might differ between humans and mice. On the
contrary, our data show that the same four transcription factors
induce iPS cells in both humans and mouse. The four factors,
however, could not induce human iPS cell colonies when fibroblasts
were kept under the culture condition for mouse ES cells after
retroviral transduction (See Example 13, above), even though these
cells stained positive for alkaline phosphatase. These data suggest
that the fundamental transcriptional network governing pluripotency
is common in human and mice, but extrinsic factors and signals
maintaining pluripotency are unique for each species.
[0197] Deciphering of the mechanism by which the four factors
induce pluripotency in somatic cells remains elusive. The function
of Oct3/4 and Sox2 as core transcription factors to determine
pluripotency is well documented (Boyer et al., Cell 122:947-956,
2005; Loh et al., Nat Genet 38:431-440, 2006; Wang et al., Nature
444:364-368, 2006). They synergistically upregulate "stemness"
genes, while suppressing differentiation-associated genes in both
mouse and human ES cells. However, they cannot bind their targets
genes in differentiated cells, because of other inhibitory
mechanisms, including DNA methylation. It may be speculated that
c-Myc and Klf4 modifies chromatin structure so that Oct3/4 and Sox2
can bind to their targets (Yamanaka, Cell Stem Cell 1:39-49, 2007).
Notably, Klf4 interacts with p300 histone acetyltransferase and
regulates gene transcription by modulating histone acetylation
(Evans et al., J Biol Chem, 2007).
[0198] The negative role of c-Myc in the self renewal of hES cells
has also been reported (Sumi et al., Oncogene 26: 5564-5576, 2007).
They showed that forced expression of c-Myc induced differentiation
and apoptosis of human ES cells. During iPS cell generation,
transgenes derived from retroviruses are silenced when the
transduced fibroblasts acquire ES-like state. The role of c-Myc in
establishing iPS cells may be as a booster of reprogramming, rather
than a controller of maintenance of pluripotency.
[0199] It has been found that each iPS clone contained 3-6
retroviral integrations for each factor. Thus, each clone had more
than 20 retroviral integration sites in total, which may increase
the risk of tumorigenesis. In the case of mouse iPS cells,
.about.20% of chimera mice and their offspring derived from iPS
cells developed tumors Okita et al., Nature 448:313-17, 2007). This
issue must be overcome to use iPS cells in human therapies.
Therefore, non-retroviral methods to introduce the four factors,
such as adenoviruses or cell-permeable recombinant proteins, are
also contemplated as part of the invention. Alternatively, small
molecules may replace the four factors for the induction of iPS
cells.
[0200] Experimental Procedures for Examples 14-20.
[0201] Cell Culture
[0202] HDFs from facial dermis of 36-year-old Caucasian female and
HFLS from synovial tissue of 69-year-old Caucasian male were
purchased from Cell Applications, Inc. BJ fibroblasts from neonatal
foreskin and NTERA-2 clone D1 human embryonic carcinoma cells were
obtained from American Type Culture Collection. Human fibroblasts,
NTERA-2, PLAT-E, and PLAT-A cells were maintained in Dulbecco's
modified eagle medium (DMEM, Nacalai Tesque, Japan) containing 10%
fetal bovine serum (FBS, Japan Serum) and 0.5% penicillin and
streptomycin (Invitrogen). 293FT cells were maintained in DMEM
containing 10% FBS, 2 mM L-glutamine (Invitrogen),
1.times.10.sup.-4 M nonessential amino acids (Invitrogen), 1 mM
sodium pyruvate (Sigma) and 0.5% penicillin and streptomycin. PA6
stroma cells (RIKEN Bioresource Center, Japan) were maintained in
.alpha.-MEM containing 10% FBS and 0.5% penicillin and
streptomycin. iPS cells were generated and maintained in Primate ES
medium (ReproCELL, Japan) supplemented with 4 ng/ml recombinant
human basic fibroblast growth factor (bFGF, WAKO, Japan). For
passaging, human iPS cells were washed once with PBS and then
incubated with DMEM/F12 containing 1 mg/ml collagenase IV
(Invitrogen) at 37.degree. C. When colonies at the edge of the dish
started dissociating from the bottom, DMEF/F12/collangenase was
removed and washed with hES cell medium. Cells were added, and the
contents were transferred to a new dish on SNL feeder cells. The
split ratio was routinely 1:3. For feeder-free culture of iPS
cells, the plate was coated with 0.3 mg/ml Matrigel (growth-factor
reduced, BD Biosciences) at 4.degree. C. overnight. The plate was
warmed to room temperature before use. Unbound Matrigel was
aspirated off and washed out with DMEM/F12. iPS cells were seeded
on Matrigel-coated plate in MEF-CM or ES medium, both supplemented
with 4 ng/ml bFGF. The medium was changed daily. For preparation of
MEF-CM, MEFs derived from embryonic day 13.5 embryo pool of ICR
mice were plated at 1.times.10.sup.6 cells per 100 mm dish and
incubated overnight. Next day, the cells were washed once with PBS
and cultured in 10 ml of ES medium. Twenty-four h after incubation,
the supernatant of MEF culture was collected, filtered through a
0.22 .mu.m pore-size filter, and stored at -20.degree. C. until
use.
[0203] Plasmid Construction
[0204] The open reading frame of human OCT3/4 was amplified by
RT-PCR and cloned into pCR2.1-TOPO. An EcoRI fragment of
pCR2.1-hOCT3/4 was introduced into the EcoRI site of pMXs
retroviral vector. To discriminate each experiment, a 20-bp random
sequence, designated N.sub.20 barcode, was introduced into the
NotI/SalI site of Oct3/4 expression vector. A unique barcode
sequence was used in each experiment to avoid inter-experimental
contamination. The open reading frames of human SOX2, KLF4, and
c-MYC were also amplified by RT-PCR and subcloned into pENTR-D-TOPO
(Invitrogen). All of the genes subcloned into pENTR-D-TOPO were
transferred to pMXs by using the Gateway cloning system
(Invitrogen), according to the manufacturer's instructions. Mouse
Slc7a1 ORF was also amplified, subcloned into pENTR-D-TOPO, and
transferred to pLenti6/UbC/V5-DEST (Invitrogen) by the Gateway
system. The regulatory regions of the human OCT3/4 gene and the
REX1 gene were amplified by PCR and subcloned into pCRXL-TOPO
(Invitrogen). For phOCT4-Luc and phREX1-Luc, the fragments were
removed by KpnI/BglII digestion from pCRXL vector and subcloned
into the KpnI/BglII site of pGV-BM2. For pPolII-Luc, an AatII
(blunted)/NheI fragment of pQBI-polII was inserted into the KpnI
(blunted)/NheI site of pGV-BM2. All of the fragments were verified
by sequencing. Primer sequences are shown in TABLE 13.
TABLE-US-00013 TABLE 13 Primer Sequences SEQ ID Primer NO: Sequence
(5' to 3') Applications hOCT3/4-S944 26 CCC CAG GGC CCC ATT TTG GTA
CC OCT3/4 Tg PCR hSOX2-S691 27 GGC ACG CCT GGG ATG GCT CTT GGC TC
SOX2 Tg PCR hKLF4-S1128 28 ACG ATC GTG GCC CCG GAA AAG GAC C KLF4
endo and Tg PCR hMYC-S1011 29 CAA CAA CCG AAA ATG CAC CAG CCC c-MYC
Tg PCR CAG pMXs-AS3200 30 TTA TCG TCG ACC ACT GTG CTG CTG Tg PCR
pMXs-L3205 31 CCC TTT TTC TGG AGA CTA AAT AAA Tg PCR hOCT3/4-S1165
32 GAC AGG GGG AGG GGA GGA GCT AGG Endo OCT3/4 hOCT3/4-AS1283 33
CTT CCC TCC AAC CAG TTG CCC CAA AC RT-PCR hSOX2-S1430 34 GGG AAA
TGG GAG GGG TGC AAA AGA GG Endo SOX2 hSOX2-AS1555 35 TTG CGT GAG
TGT GGA TGG GAT TGG TG RT-PCR ECAT4-macaca-968S 36 CAG CCC CGA TTC
TTC CAC CAG TCC C NANOG RT-PCR ECAT4-macaca- 37 CGG AAG ATT CCC AGT
CGG GTT CAC C 1334AS hGDF3-S243 38 CTT ATG CTA CGT AAA GGA GCT GGG
GDF3 RT-PCR hGDF3-AS850 39 GTG CCA ACC CAG GTC CCG GAA GTT
hREXI-RT-U 40 CAG ATC CTA AAC AGC TCG CAG AAT REX1 RT-PCR
hREXI-RT-L 41 GCG TAC GCA AAT TAA AGT CCA GA hFGF4-RT-U 42 CTA CAA
CGC CTA CGA GTC CTA CA FGF4 RT-PCR hFGF4-RT-L 43 GTT GCA CCA GAA
AAG TCA GAG TTG hpH34-S40 44 ATA TCC CGC CGT GGG TGA AAG TTC ESG1
RT-PCR hpH34-AS259 45 ACT CAG CCA TGG ACT GGA GCA TCC
hECAT15-1-S532 46 GGA GCC GCC TGC CCT GGA AAA TTC DPPA4 RT-PCR
hECAT15-1-AS916 47 TTT TTC CTG ATA TTC TAT TCC CAT bECAT15-2-S85 48
CCG TCC CCG CAA TCT CCT TCC ATC DPPA2 RT-PCR hECAT15-2-AS667 49 ATG
ATG CCA ACA TGG CTC CCG GTG hTERT-S3234 50 CCT GCT CAA GCT GAC TCG
ACA CCG TG hTERT RT-PCR hTERT-AS3713 51 GGA AAA GCT GGC CCT GGG GTG
GAG C hKLF4-AS1826 52 TGA TTG TAG TGC TTT CTG GCT GGG CTC Endo KLF4
C RT-PCR hMYC-S253 53 GCG TCC TGG GAA GGG AGA TCC GGA GC Endo c-MYC
hMYC-AS555 54 TTG AGG GGC ATC GTC GCG GGA GGC TG RT-PCR hMSX1-S665
55 CGA GAG GAC CCC GTG GAT GCA GAG MSX1 RT-PCR hMSX1-AS938 56 GGC
GGC CAT CTT CAG CTT CTC CAG hBRACHYURY- 57 GCC CTC TCC CTC CCC TCC
ACG CAC AG BRACHYURY/T S1292 RT-PCR hBRACHYURY- 58 CGG CGC CGT TGC
TCA CAG ACC ACA GG AS1540 hGFAP-S1040 59 GGC CCG CCA CTT GCA GGA
GTA CCA GG GFAP RT-PCR hGFAP-AS1342 60 CTT CTG CTC GGG CCC CTC ATG
AGA CG hPAX6-S1206 61 ACC CAT TAT CCA GAT GTG TTT GCC CGA PAX6
RT-PCR G hPAX6-AS1497 62 ATG GTG AAG CTG GGC ATA GGC GGC AG
hFOXA2-S208 63 TGG GAG CGG TGA AGA TGG AAG GGC AC FOXA2 RT-PCR
hFOXA2-AS398 64 TCA TGC CAG CGC CCA CGT ACG ACG AC hSOX17-S423 65
CGC TTT CAT GGT GTG GGC TAA GGA CG SOX17 RT-PCR hSOX17-AS583 66 TAG
TTG GGG TGG TCC TGC ATG TGC TG hAADC-S1378 67 CGC CAG GAT CCC CGC
TTT GAA ATC TG AADC RT-PCR hAADC-AS1594 68 TCG GCC GCC AGC TCT TTG
ATG TGT TC hChAT-S1360 69 GGA GGC GTG GAG CTC AGC GAC ACC ChAT
RT-PCR hChAT-AS1592 70 CGG GGA GCT CGC TGA CGG AGT CTG hMAP2-S5401
71 CAG GTG GCG GAC GTG TGA AAA TTG MAP2 RT-PCR AGA GTG hMAP2-AS5587
72 CAC GCT GGA TCT GCC TGG GGA CTG TG hDAT-S 1935 73 ACA GAG GGG
AGG TGC GCC AGT TCA CG hDAT-AS2207 74 ACG GGG TGG ACC TCG CTG CAC
AGA TC SLC6A3/DAT RT-PCR hLMX1B-S770 75 GGC ACC AGG AGC AGC AGG AGC
AGC AG hLMXIB-AS1020 76 CCA CGT CTG AGG AGC CGA GGA AGC AG LMX1B
RT-PCR bMYL2A-S258 77 GGG CCC CAT CAA CTT CAC CGT CTT CC
hMYL2A-AS468 78 TGT AGT CGA TGT TCC CCG CCA GGT CC MYL2A RT-PCR
hTnTc-S524 79 ATG AGC GGG AGA AGG AGC GGC AGA AC hTnTc-AS730 80 TCA
ATG GCC AGC ACC TTC CTC CTC TC TnTc RT-PCR hMEF2C-S1407 81 TTT AAC
ACC GCC AGC CGT CTT CAC CTT MEF2C RT-PCR G hMEF2C-AS1618 82 TCG TGG
CGC GTG TGT TGT GGG TAT CTC G hMYHCB-S5582 83 CTG GAG GCC GAG CAG
AAG CGC AAC G MYHCB RT-PCR hMYHCB-AS5815 84 GTC CGC CCG CTC CTC TGC
CTC ATC C dT.sub.20 85 TTT TTT TTT TTT TTT TTT TT Reverse
transcription bMYC-S857 86 GCC ACA GCA AAC CTC CTC ACA GCC CAC
Southern blot probe bMYC-AS1246 87 CTC GTC GTT TCC GCA ACA AGT CCT
CTT C hOCT3/4-S 88 CAC CAT GGC GGG ACA CCT GGC TTC AG OCT3/4
cloning hOCT3/4-AS 89 ACC TCA GTT TGA ATG CAT GGG AGA GC hSOX2-S 90
CAC CAT GTA CAA CAT GAT GGA GAC SOX2 cloning GGA GCT G hSOX2-AS 91
TCA CAT GTG TGA GAG GGG CAG TGT GC hKLF4-S 92 CAC CAT GGC TGT CAG
TGA CGC GCT GCT KLF4 cloning CCC hKLF4-AS 93 TTA AAA ATG TCT CTT
CAT GTG TAA GGC GAG hMYC-S 94 CAC CAT GCC CCT CAA CGT TAG CTT CAC
c-MYC cloning CAA hMYC-AS 95 TCA CGC ACA AGA GTT CCG TAG CTG TTC
AAG Slc7a1-S 96 CAC CAT GGG CTG CAA AAA CCT GCT CGG Mouse Slc7a1
Slc7a1-AS 97 TCA TTT GCA CTG GTC CAA GTT GCT GTC cloning
hREX1-pro5K-S 98 ATT GTC GAC GGG GAT TTG GCA GGG TCA CAG GAC
hREXx1-pro5K-AS 99 CCC AGA TCT CCA ATG CCA CCT CCT CCC Promoter
cloning AAA CG hOCT3/4-pro5K-S 100 CACTCG AGG TGG AGG AGC TGA GGG
CAC TGT GG hOCT3/4-pro5K-AS 101 CAC AGA TCT GAA ATG AGG GCT TGC GAA
GGG AC mehREX1-F1-S 102 GGT TTA AAA GGG TAA ATG TGA TTA TAT
Bisulfite sequencing TTA mehREX1-F1-AS 103 CAA ACT ACA ACC ACC CAT
CAA C mehOCT3/4 F2-S 104 GAG GTT GGA GTA GAA GGA TTG TTT TGG TTT
mehOCT3/4 F2-AS 105 CCC CCC TAA CCC ATC ACC TCC ACC ACC TAA
mehNANOG-FI-S 106 TGG TTA GGT TGG TTT TAA ATT TTT G mehNANOG-FI-AS
107 AAC CCA CCC TTA TAA ATT CTC AAT TA
[0205] Lentivirus Production and Infection
[0206] 293FT cells (Invitrogen) were plated at 6.times.10.sup.6
cells per 100 mm dish, and incubated overnight. Cells were
transfected with 3 .mu.g of pLenti6/UbC-Slc7a1 along with 9 .mu.g
of Virapower packaging mix by Lipofectamine 2000 (Invitrogen),
according to the manufacturer's instructions. Forty-eight hours
after transfection, the supernatant of transfectant was collected
and filtered through a 0.45 .mu.m pore-size cellulose acetate
filter (Whatman). Human fibroblasts were seeded at 8.times.10.sup.5
cells per 100 mm dish 1 day before transduction. The medium was
replaced with virus-containing supernatant supplemented with 4
.mu.g/ml polybrene (Nacalai Tesque), and incubated for 24
hours.
[0207] Retroviral Infection and iPS Cell Generation
[0208] PLAT-E packaging cells were plated at 8.times.10.sup.6 cells
per 100 mm dish and incubated overnight. Next day, the cells were
transfected with pMXs vectors with Fugene 6 transfection reagent
(Roche). Twenty-four hours after transfection, the medium was
collected as the first virus-containing supernatant and replaced
with a new medium, which was collected after 24 hours as the second
virus-containing supernatant. Human fibroblasts expressing mouse
Slc7a1 gene were seeded at 8.times.10.sup.5 cells per 100 mm dish 1
day before transduction. The virus-containing supernatants were
filtered through a 0.45-.mu.m pore-size filter, and supplemented
with 4 .mu.g/ml polybrene. Equal amounts of supernatants containing
each of the four retroviruses were mixed, transferred to the
fibroblast dish, and incubated overnight. Twenty-four hours after
transduction, the virus-containing medium was replaced with the
second supernatant. Six days after transduction, fibroblasts were
harvested by trypsinization and re-plated at 5.times.10.sup.4 cells
per 100-mm dish on an SNL feeder layer. Next day, the medium was
replaced with hES medium supplemented with 4 ng/ml bFGF. The medium
was changed every other day. Thirty days after transduction,
colonies were picked up and transferred into 0.2 ml of hES cell
medium. The colonies were mechanically dissociated to small clumps
by pipeting up and down. The cell suspension was transferred on SNL
feeder in 24-well plates. This stage was defined as passage 1.
[0209] RNA Isolation and Reverse Transcription
[0210] Total RNA was purified with Trizol reagent (Invitrogen) and
treated with Turbo DNA-free kit (Ambion) to remove genomic DNA
contamination. One microgram of total RNA was used for reverse
transcription reaction with ReverTraAce-.alpha. (Toyobo, Japan) and
dT.sub.20 primer, according to the manufacturer's instructions. PCR
was performed with ExTaq (Takara, Japan). Quantitative PCR was
performed with Platinum SYBR Green qPCR Supermix UDG (Invitrogen)
and analyzed with the 7300 real-time PCR system (Applied
Biosystems). Primer sequences are shown in TABLE 13.
[0211] Alkaline Phosphatase Staining and Immunocytochemistry
[0212] Alkaline phosphatase staining was performed using the
Leukocyte Alkaline Phosphatase kit (Sigma). For
immunocytochemistry, cells were fixed with PBS containing 4%
paraformaldehyde for 10 min at room temperature. After washing with
PBS, the cells were treated with PBS containing 5% normal goat or
donkey serum (Chemicon), 1% bovine serum albumin (BSA, Nacalai
tesque), and 0.1% Triton X-100 for 45 min at room temperature.
Primary antibodies included SSEA1 (1:100, Developmental Studies
Hybridoma Bank), SSEA3 (1:10, a kind gift from Dr. Peter W.
Andrews), SSEA4 (1:100, Developmental Studies Hybridoma Bank),
TRA-2-49/6E (1:20, Developmental Studies Hybridoma Bank), TRA-1-60
(1:50, a kind gift from Dr. Peter W. Andrews), TRA-1-81 (1:50, a
kind gift from Dr. Peter W. Andrews), Nanog (1:20, AF1997, R&D
Systems), .beta.III-tubulin (1:100, CB412, Chemicon), glial
fibrillary acidic protein (1:500, Z0334, DAKO), .alpha.-smooth
muscle actin (pre-diluted, N1584, DAKO), desmin (1:100, RB-9014,
Lab Vision), vimentin (1:100, SC-6260, Santa Cruz),
.alpha.-fetoprotein (1:100, MAB1368, R&D Systems), tyrosine
hydroxylase (1:100, AB152, Chemicon). Secondary antibodies used
were cyanine 3 (Cy3)-conjugated goat anti-rat IgM (1:500, Jackson
Immunoresearch), Alexa546-conjugated goat anti-mouse IgM (1:500,
Invitrogen), Alexa488-conjugated goat anti-rabbit IgG (1:500,
Invitrogen), Alexa488-conjugated donkey anti-goat IgG (1:500,
Invitrogen), Cy3-conjugated goat anti-mouse IgG (1:500, Chemicon),
and Alexa488-conjugated goat anti-mouse IgG (1:500, Invitrogen).
Nuclei were stained with 1 .mu.g/ml Hoechst 33342 (Invitrogen).
[0213] In Vitro Differentiation
[0214] For EB formation, human iPS cells were harvested by treating
with collagenase IV. The clumps of the cells were transferred to
poly(2-hydroxyethyl methacrylate)-coated dish in DMEM/F12
containing 20% knockout serum replacement (KSR, Invitrogen), 2 mM
L-glutamine, 1.times.10.sup.-4 M nonessential amino acids,
1.times.10.sup.-4 M 2-mercaptoethanol (Invitrogen), and 0.5%
penicillin and streptomycin. The medium was changed every other
day. After 8 days as a floating culture, EBs were transferred to
gelatin-coated plate and cultured in the same medium for another 8
days. Co-culture with PA6 was used for differentiation into
dopaminergic neurons. PA6 cells were plated on gelatin-coated
6-well plates and incubated for 4 days to reach confluence. Small
clumps of iPS cells were plated on PA6-feeder layer in Glasgow
minimum essential medium (Invitrogen) containing 10% KSR
(Invitrogen), 1.times.10.sup.-4 M nonessential amino acids,
1.times.10.sup.-4 M 2-mercaptoethanol (Invitrogen), and 0.5%
penicillin and streptomycin. For cardiomyocyte differentiation, iPS
cells were maintained on Matrigel-coated plate in MEF-CM
supplemented with 4 ng/ml bFGF for 6 days. The medium was then
replaced with RPMI1640 (Invitrogen) plus B27 supplement
(Invitrogen) medium (RPMI/B27), supplemented with 100 ng/ml human
recombinant activin A (R & D Systems) for 24 hours, followed by
10 ng/ml human recombinant bone morphologenic protein 4 (BMP4,
R&D Systems) for 4 days. After cytokine stimulation, the cells
were maintained in RPMI/B27 without any cytokines. The medium was
changed every other day.
[0215] Bisulfite Sequencing
[0216] Genomic DNA (1 .mu.g) was treated with CpGenome DNA
modification kit (Chemicon), according to the manufacturer's
recommendations. Treated DNA was purified with QIAquick column
(QIAGEN). The promoter regions of the human Oct3/4, Nanog, and Rex1
genes were amplified by PCR. The PCR products were subcloned into
pCR2.1-TOPO. Ten clones of each sample were verified by sequencing
with the M13 universal primer. Primer sequences used for PCR
amplification were provided in TABLE 13.
[0217] Luciferase Assay
[0218] Each reporter plasmid (1 .mu.g) containing the firefly
luciferase gene was introduced into human iPS cells or HDF with 50
ng of pRL-TK (Promega). Forty-eight hours after transfection, the
cells were lysed with 1.times. passive lysis buffer (Promega) and
incubated for 15 min at room temperature. Luciferase activities
were measured with a Dual-Luciferase reporter assay system
(Promega) and Centro LB 960 detection system (BERTHOLD), according
to the manufacturer's protocol.
[0219] Teratoma Formation
[0220] The cells were harvested by collagenase IV treatment,
collected into tubes and centrifuged, and the pellets were
suspended in DMEM/F12. One quarter of the cells from a confluent
100 mm dish was injected subcutaneously to dorsal flank of a SCID
mouse (CREA, Japan). Nine weeks after injection, tumors were
dissected, weighted, and fixed with PBS containing 4%
paraformaldehyde. Paraffin-embedded tissue was sliced and stained
with hematoxylin and eosin.
[0221] Western Blotting
[0222] The cells at semiconfluent state were lysed with RIPA buffer
(50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40 (NP-40), 1%
sodium deoxycholate, and 0.1% SDS), supplemented with protease
inhibitor cocktail (Roche). The cell lysate of MEL-1 hES cell line
was purchased from Abcam. Cell lysates (20 .mu.g) were separated by
electrophoresis on 8% or 12% SDS-polyacrylamide gel and transferred
to a polyvinylidine difluoride membrane (Millipore). The blot was
blocked with TBST (20 mM Tris-HCl, pH 7.6, 136 mM NaCl, and 0.1%
Tween-20) containing 1% skim milk and then incubated with primary
antibody solution at 4.degree. C. overnight. After washing with
TBST, the membrane was incubated with horseradish peroxidase
(HRP)-conjugated secondary antibody for 1 hour at room temperature.
Signals were detected with Immobilon Western chemiluminescent HRP
substrate (Millipore) and LAS3000 imaging system (FUJIFILM, Japan).
Antibodies used for western blotting were anti-Oct3/4 (1:600,
SC-5279, Santa Cruz), anti-Sox2 (1:2000, AB5603, Chemicon),
anti-Nanog (1:200, R&D Systems), anti-Klf4 (1:200, SC-20691,
Santa Cruz), anti-c-Myc (1:200, SC-764, Santa Cruz),
anti-E-cadherin (1:1000, 610182, BD Biosciences), anti-Dppa4
(1:500, ab31648, Abeam), anti-FoxD3 (1:200, AB5687, Chemicon),
anti-telomerase (1:1000, ab23699, Abeam), anti-Sall4 (1:400,
ab29112, Abeam), anti-LIN-28 (1:500, AF3757, R&D systems),
anti-.beta.-actin (1:5000, A5441, Sigma), anti-mouse IgG-HRP
(1:3000, #7076, Cell Signaling), anti-rabbit IgG-HRP (1:2000,
#7074, Cell Signaling), and anti-goat IgG-HRP (1:3000, SC-2056,
Santa Cruz).
[0223] Southern Blotting
[0224] Genomic DNA (5 .mu.g) was digested with BglII, EcoRI, and
NcoI overnight. Digested DNA fragments were separated on 0.8%
agarose gel and transferred to a nylon membrane (Amersham). The
membrane was incubated with digoxigenin (DIG)-labeled DNA probe in
DIG Easy Hyb buffer (Roche) at 42.degree. C. overnight with
constant agitation. After washing, alkaline phosphatase-conjugated
anti-DIG antibody (1:10,000, Roche) was added to a membrane.
Signals were raised by CDP-star (Roche) and detected by LAS3000
imaging system.
[0225] Short Tandem Repeat Analysis and Karyotyping
[0226] The genomic DNA was used for PCR with Powerplex 16 system
(Promega) and analyzed by ABI PRISM 3100 Genetic analyzer and Gene
Mapper v3.5 (Applied Biosystems). Chromosomal G-band analyses were
performed at the Nihon Gene Research Laboratories, Japan.
[0227] Detection of Telomerase Activity
[0228] Telomerase activity was detected with a TRAPEZE telomerase
detection kit (Chemicon), according to the manufacturer's
instructions. The samples were separated by TBE-based 10%
acrylamide non-denaturing gel electrophoresis. The gel was stained
with SYBR Gold (1:10,000, Invitrogen).
Example 21
Generation of Induced Pluripotent Stem Cells without Myc
[0229] The direct reprogramming of somatic cells is considered to
provide an opportunity to generate patient- or disease-specific
pluripotent stem cells. Such pluripotent stem (iPS) cells are
induced from mouse fibroblasts by the retroviral transduction of
four transcription factors, Oct3/4, Sox2, Klf4, and c-Myc
(Takahashi et al., Cell 126:663-76, 2006). Mouse iPS cells are
indistinguishable from mouse ES cells in many aspects and give rise
to germline-competent chimeras (Wernig et al., Nature 448:318-24,
2007; Okita et al., Nature 448:313-17, 2007; Maherali et al., Cell
Stem Cell 1:55-70, 2007). It was noted above that each iPS clone
contained 3-6 retroviral integrations for each factor. Thus, each
clone had more than 20 retroviral integration sites in total, which
may increase the risk of tumorigenesis. In the case of mouse iPS
cells, .about.20% of chimera mice and their offspring derived from
iPS cells developed tumors (Okita et al., Nature 448:313-17, 2007).
In particular, it was found that the reactivation of the c-Myc
retrovirus results in an increased incidence of tumor formation in
the chimeras and progeny mice generated with mouse iPS cells, thus
hindering the clinical application of this technology (Okita et
al., Nature 448:313-17, 2007). Therefore, a modified protocol for
the induction of iPS cells, which does not require the Myc
retrovirus was developed. With this new protocol, significantly
fewer non-iPS background cells were obtained. Furthermore, the iPS
cells generated without Myc were constantly of high quality. These
findings are important for the future clinical application of this
iPS cell technology.
[0230] Substitution of the Four Factors with Other Family
Members
[0231] This study was initiated to examine whether the family
proteins of the four factors could also induce iPS cells. In other
words, further investigations were performed to assess which family
members of a given gene family could substitute for others as
nuclear reprogramming factors. Mouse embryonic fibroblasts (MEF)
containing a GFP-IRES-Puro.sup.r transgene driven by the Nanog gene
regulatory elements were used (Okita et al., Nature 448:313-17,
2007). Nanog is specifically expressed in mouse ES cells and
preimplantation embryos (Chambers et al., Cell 113: 643-55, 2003;
Mitsui et al., Cell 113: 631-42, 2003) and can serve as a selection
marker during iPS cell induction. By introducing the aforementioned
four factors, iPS cells are induced as GFP-expressing colonies.
Nanog-selected iPS cells are indistinguishable from ES cells and
have been shown to give rise to germline-competent chimeras (Wernig
et al., Nature 448: 318-24, 2007; Okita et al., Nature 448:313-17,
2007; Maherali et al., Cell Stem Cell 1:55-70, 2007).
[0232] Oct3/4 belongs to the Oct family transcription factors,
which contain the POU domain (Ryan et al., Genes Dev 11: 1207-25,
1997). The closest homologs of Oct3/4 are Oct1 and Oct6. Oct3/4,
Oct1, or Oct6 were introduced together with the remaining three
factors, into the Nanog-reporter MEF by retroviruses. With Oct3/4,
many GFP-positive colonies were observed (FIG. 43A). In contrast,
no GFP-positive colonies were obtained with Oct1 or Oct6, thus
indicating the inability of these two homologs to induce iPS
cells.
[0233] Sox2 belongs to the Sox (SRY-related HMG-box) transcription
factors, characterized by the presence of the high mobility group
(HMG) domain (Schepers et al., Dev Cell 3: 167-70, 2002). Sox1,
Sox3, Sox7, Sox15, Sox17, and Sox18 were tested and GFP-positive
colonies were obtained with Sox1. In addition, fewer GFP-positive
colonies were obtained with Sox3, Sox15, and Sox18 (FIG. 43A).
Sox18, however, failed to expand the cells.
[0234] Klf4 belongs to Kruppel-like factors (Klfs), zinc-finger
proteins that contain amino acid sequences that resemble those of
the Drosophila embryonic pattern regulator Kruppel (Dang et al.,
Int J Biochem Cell Biol 32: 1103-21, 2000). Klf1, Klf2, and Klf5
were tested and GFP-expressing colonies with Klf2 were thus
obtained (FIG. 43A). Klf1 and Klf5 were also capable of inducing
iPS cells, but with a lower efficiency.
[0235] c-Myc has two related proteins, N-Myc and L-Myc (Adhikary et
al., Nat Rev Mol Cell Biol 6: 635-45, 2005). GFP-positive colonies
emerged with both N-Myc and L-Myc (FIG. 43A). Therefore, some, but
not all family proteins of the four factors can induce iPS
cells.
[0236] The family proteins were also tested for their ability to
induce iPS cells from MEFs in which .beta.geo was knocked into the
Fbx15 locus (Tokuzawa et al., Mol Cell Biol 23: 2699-708, 2003).
Similar results to those with the Nanog-based selection were
obtained: Sox2 could be replaced by Sox1 and Sox3, Klf4 by Klf2,
and c-Myc by N-Myc and L-Myc. The cells generated by the family
proteins were expandable and showed a morphology indistinguishable
from that of ES cells (not shown). They gave rise to teratomas in
nude mice (FIG. 44). Therefore, some family proteins are capable of
inducing iPS cells from both Nanog-reporter MEF and Fbx15-reporter
MEF.
[0237] As has been stated above, iPS cell generation from somatic
cells was evaluated by optimizing retroviral transduction and
subsequent culture conditions. Furthermore, optimization would be
useful for the application of this iPS cell technology to human
cells, especially in clinical situations. Unexpectedly, a few ES
cell-like and GFP-positive colonies from Nanog-reporter MEF were
obtained without any Myc retroviruses (FIG. 43A). This was in
contrast to a previous study in which no GFP-positive colonies
could be obtained without c-Myc (Okita et al., Nature 448:313-17,
2007). Consistent with the efforts toward optimization, one
difference between the two studies is the timing of the drug
selection: In the previous study, puromycin selection was initiated
seven days after the transduction, whereas in this experiment the
selection was started at 14 days. This suggests that iPS cell
induction without Myc is a slower process than that with Myc.
Furthermore, as is further discussed herein, the omission of Myc
resulted in a less efficient but more specific induction of iPS
cells.
[0238] Myc Omission Results in More Specific iPS Cell Induction
[0239] To test whether iPS cell induction without Myc is a slower
process than that with Myc, Nanog-reporter MEFs were transduced
with either the four factors or three factors devoid of Myc, and
then puromycin selection was started seven, 14, or 21 days after
the transduction (FIG. 43B). With the four factors, GFP-positive
colonies were observed in all of the conditions. The colony numbers
significantly increased when puromycin selection was delayed.
Without Myc, no GFP-colonies were observed when selection was
initiated seven days after the transduction. In contrast,
GFP-positive colonies did emerge even without Myc when selection
was started 14 or 21 days after the transduction. The colony
numbers were fewer with the three factors than with the four
factors in each condition. Nanog-selected iPS cells generated
without Myc retroviruses expressed ES cell marker genes at
comparable levels to those in ES cells (FIG. 45), and thus gave
rise to adult chimeras when transplanted into blastocysts (TABLE
14).
TABLE-US-00014 TABLE 14 Summary of blastocysts injections iPS
origin- injected clones genotype* selection blastocysts born mice
chimeras 142B-6 MEF-FB/gfp G418 39 7 3 142B-12 46 12 5 178B-1
MEF-Ng Puro 156 50 5 178B-2 142 43 17 178B-5 60 20 5 178B-6 28 10 4
256H-4 TTF-ACTB- No 72 6 5 256H-13 DsRed 96 8 5 256H-18 90 17 11
All iPS clones were induced with three factors devoid of Myc from
MEF or TTF. *FB, Fbx15-.beta.geo reporter; Ng,
Nanog-GFP-IRES-Puro.sup.r reporter; gfp, CAG-EGFP
[0240] Another difference is that fewer GFP-negative colonies as
well as background cells were observed with the three factors
devoid of Myc than with the four factors (FIG. 43C). Therefore, the
omission of Myc resulted in a less efficient but more specific
induction of iPS cells.
[0241] It was also possible to generate a few iPS cells without Myc
from MEFs, in which .beta.geo was knocked into the Fbx15 locus
(Tokuzawa et al., Mol. Cell Biol. 23:2699-708, 2003). (FIG. 46A).
This is again in contrast to the original report, in which no iPS
cells were obtained without c-Myc (Takahashi et al., Cell
126:663-76, 2006). In the two experiments, G418 selection was
initiated with the same timing: three days after the transduction.
However, the colonies were selected 14-21 days after the
transduction in the previous report, whereas .about.30 days were
required in the current study. Another difference was that the
retroviral transfection efficiency was raised by preparing each of
the four or three factors separately in an independent Plat-E
(Morita et al., Gene Ther. 7:1063-66, 2000) plate in this study. In
comparison to the original work in which all the four factors were
prepared in a single Plat-E plate, a significant increase in the
number of iPS cell colonies was observed (not shown). This is
consistent with the notion that iPS cell induction without Myc is a
slower and less efficient process than that with Myc.
[0242] Fbx15-selected iPS cells, which were generated with the four
factors, express lower levels of ES-cell marker genes than ES cells
(Takahashi et al., Cell 126:663-76, 2006). They cannot produce
adult chimeras when microinjected into blastocysts. In contrast,
iPS cells generated without Myc expressed ES-cell marker genes at
comparable levels to those in ES cells even with the Fbx15
selection (FIG. 46B). Furthermore, adult chimeras were obtained
with high iPS cell contribution from these cells (FIG. 46C, TABLE
14). No increased incidence of tumor formation was observed in
these chimeras.
[0243] iPS Cell Induction in the Absence of Drug Selection
[0244] Next, it was determined whether the omission of Myc would
result in efficient isolation of iPS cells without drug selection.
The four or three factors were introduced into adult tail tip
fibroblasts (TTF) containing the Nanog reporter, but puromycin
selection was not applied. DsRed retrovirus was transduced together
with the four or three factors to visualize transduced cells.
Thirty days after the retroviral transduction, the dishes
transduced with the four factors were covered with numerous
GFP-negative colonies and background cells (FIG. 47A, TABLE
15).
TABLE-US-00015 TABLE 15 Summary of experiments (Nanog-GFP reporter
TTF, without selection) Experiment Cell Total picked Number Factors
seeded colonies up established 220 4 5 .times. 10.sup.4 many (107)
26 (24) 25 (22) 256 4 5 .times. 10.sup.4 many (4) 3 3.5 .times.
10.sup.5 7 (4) 7 (4) 6 (5) 272 4 5.4 .times. 10.sup.4 many (132) 6
(6) 5 (4) 3 3.1 .times. 10.sup.5 21 (8) 4 (4) 2 (2) 309 4 2.3
.times. 10.sup.4 many (424) 3 9.6 .times. 10.sup.5 43 (24) Numbers
in parentheses indicate number of colonies or clones that were
positive for GFP. The ratios of the retroviruses, Oct3/4, Sox2,
Klf4, (c-Myc), and DsRed, were 1:1:1:(1):4 in experiment 256 and
1:1:1:(1):1 in experiments 272 and 309. In experiment 220, DsRed
was not introduced.
[0245] Using fluorescent microscopy, small portions of these
colonies (4, 132, and 424 colonies in three independent
experiments) were found GFP-positive. Of note, the GFP-positive
colonies were negative for DsRed, which was consistent with the
retroviral silencing observed in Nanog-selected iPS cells (Okita et
al., Nature 448:313-17, 2007). In contrast, with the three factors
devoid of Myc, a small number (7, 21, and 43 in three independent
experiments) of discrete colonies were observed with few background
cells. Approximately a half of them expressed GFP in a patchy
manner. DsRed was only detected in a small portion of some
colonies, indicating that it was largely silenced. No overlap was
observed between GFP and DsRed. Most of these colonies were
expandable and produced iPS cells, which became positive for GFP
and negative for DsRed at passage 2. Therefore, the omission of
c-Myc resulted in more specific generation of iPS cells, in which
Nanog-GFP is activated whereas the retroviruses are silenced.
[0246] Next, generation of iPS cells was attempted from adult TTF
that did not have selection markers, but had the DsRed transgene
driven by a constitutively active promoter (Vintersten et al.
Genesis 40:241-46, 2004). The four factors or the three factors
devoid of Myc were introduced. In addition, a GFP retrovirus was
introduced to monitor silencing. After 30 days without drug
selection, .about.1000 colonies emerged from 0.5.times.10.sup.5
cells transduced with the four factors. Most of them were positive
for GFP, indicating that retroviral silencing did not take place in
these cells. In contrast, only 16 colonies (FIG. 47B) emerged from
3.5.times.10.sup.5 cells transduced with the three factors devoid
of Myc. Most of these colonies express no GFP, while the remaining
expressed GFP in small portions. All of these colonies were
expandable and showed iPS- or ES-like morphology at the second
passage. They were all negative for GFP, thus indicating retroviral
silencing. RT-PCR showed that these cells expressed ES cell marker
genes at comparable levels to those in ES cells (FIG. 47C). In
addition, RT-PCR confirmed the retroviral silencing of Klf4 and the
absence of the Myc transgene in iPS cells generated with the three
factors. Furthermore, when transplanted into blastocysts, these
cells gave rise to chimeras (FIG. 47D, TABLE 14). Therefore, by
omitting Myc, good iPS cells can be efficiently generated from
adult TTF without drug selection. These findings should be useful
for the application of this iPS cell technology to human cells,
especially in clinical situations.
[0247] Induction of Human iPS Cells without Myc Retroviruses
[0248] FIGS. 48(A)-(C) show induction of human iPS cells without
Myc retroviruses. The retroviruses for Oct3/4, Sox2 and Klf4 were
introduced into BJ fibroblasts (246G) or HDF (253G). After 30 days,
a few hES cell-like colonies emerged. These cells were expandable
and showed hES cell-like morphology (FIG. 48(A)). Results were
obtained for the expression of ES cell marker genes in human iPS
cells derived from HDF without Myc retroviruses (253G) or with Myc
(253F) (FIG. 48(B)), as were results for embryoid body-mediated
differentiation of human iPS cells generated without Myc
retroviruses (FIG. 48(C)).
[0249] Experimental Procedures for Example 21.
[0250] Plasmid construction. The coding regions of family genes
were amplified by RT-PCR with primers listed in TABLE 16, subcloned
into pDONR201 or pENTR-D-TOPO (Invitrogen), and recombined with
pMXs-gw by the LR reaction (Invitrogen).
TABLE-US-00016 TABLE 16 Primers used for cloning of the family
factors Genes Sequences SEQ ID NO: Sox1 CAC CAT GTA CAG CAT GAT GAT
GGA GAC CGA CCT 108 CTA GAT ATG CGT CAG GGG CAC CGT GC 109 Sox3 CAC
CAT GTA CAG CCT GCT GGA GAC TGA ACT CAA G 110 TCA GAT GTG GGT CAG
CGG CAC CGT TCC ATT 111 Sox7 CAC CTC GGC CAT GGC CTC GCT GCT GGG
112 CTC CAT TCC TCC AGC TCT ATG ACA CAC 113 Sox15 CAC CAT GGC GCT
GAC CAG CTC CTC ACA A 114 TTA AAG GTG GGT TAC TGG CAT GGG 115 Sox17
CAC CAG AGC CAT GAG CAG CCC GGA TG 116 CGT CAA ATG TCG GGG TAG TTG
CAA TA 117 Sox18 CAC CAT GCA GAG ATC GCC GCC CGG CTA CG 118 CTA GCC
TGA GAT GCA AGC ACT GTA ATA GAC 119 Oct1 CAC CAT GAA TAA TCC ATC
AGA AAC CAA T 120 GCT CTG CAC TCA GCT CAC TGT GCC 121 Oct6 CAC CAT
GGC CAC CAC CGC GCA GTA TCT G 122 GGA ACC CAG TCC GCA GGG TCA CTG
123 Klf1 CAC CAT GAG GCA GAA GAG AGA GAG GAG GC 124 TCA GAG GTG ACG
CTT CAT GTG CAG AGC TAA 125 Klf2 CAC CAT GGC GCT CAG CGA GCC TAT
CTT GCC 126 CTA CAT ATG TCG CTT CAT GTG CAA GGC CAG 127 Klf5 CAC
CAT GCC CAC GCG GGT GCT GAC CAT G 128 TCG CTC AGT TCT GGT GGC GCT
TCA 129 L-MycWT CAC CAT GGA CTT CGA CTC GTA TCA GCA CTA TTT C 130
TTA GTA GCC ACT GAG GTA CGC GAT TCT CTT 131 N- CAC CAT GCC CAG CTG
CAC CGC GTC CAC CAT 132 MycWT TTA GCA AGT CCG AGC GTG TTC GAT CT
133
[0251] Retroviral transduction. pMXs-based retroviral vectors were
transfected into Plat-E cells (Morita et al., Gene Ther. 7:1063-66,
2000) using Fugene 6 reagents (Roche) according to manufacturer's
instruction. Twenty-four hours after transfection, the medium was
replaced. After 24 hours, virus-containing supernatant were used
for retroviral infection. In a "mixed" protocol, the mixture of
plasmids for the four factors was transfected into a single dish of
Plat-E cells. In a "separate" method, each plasmid was transfected
into separate dishes of Plat-E cells. Virus-containing supernatant
was mixed prior to transduction. Significantly higher transduction
efficiency was observed with the separate method.
[0252] Induction of iPS cells with drug selection. The induction of
iPS cells was performed as previously described (Takahashi et al.,
Cell 126:663-76, 2006; Okita et al., Nature 448:313-17, 2007) with
some modifications. Briefly, MEFs, which contained either the
Nanog-GFP-IRES-Puro.sup.r reporter or the Fbx15-.beta.geo reporter,
or both, were seeded at 1.3 and 8.0.times.10.sup.5 cells/well in
6-well plates and 100 mm dish, respectively, with SNL feeder cells
(McMahon et al., Cell 62:1073-85, 1990). The transduced cells were
cultivated with ES medium containing LIF (Meiner et al., Proc.
Natl. Acad. Sci. U.S.A. 93:14041-46. (1996). Selection with G418
(300 .mu.g/ml) or puromycin (1.5 .mu.g/ml) was started as
indicated. Twenty-five to 30 days after transduction, the number of
colonies was recorded. Some colonies were then selected for
expansion.
[0253] iPS cells induction without drug selection. TTFs were
isolated from adult Nanog-reporter mice or adult DsRed-transgenic
mice (Vintersten et al., Genesis 40:241-46, 2004).
Retroviral-containing supernatant was prepared in the separated
method. For the four-factor transduction, retrovirus-containing
supernatants for Klf4, c-Myc, Oct3/4, Sox2 and DsRed, were mixed
with the ratio of 1:1:1:1:4. When the three factors were
transduced, retrovirus-containing supernatants for Klf4, Oct3/4,
Sox2, Mock, and DsRed were mixed with the ratio of 1:1:1:1:4. With
DsRed transgenic mice, the GFP retrovirus was used instead of
DsRed. For transfection, TTFs were seeded at 8.0.times.10.sup.5
cells per 100-mm dishes, which did not have feeder cells. TTFs were
incubated in the virus/polybrene-containing supernatants for 24
hours. Four days after transduction, TTFs transduced with the three
factors were reseeded at 3.5.times.10.sup.5 cells per 100-mm dishes
with SNL feeder cells and cultured with ES medium. TTFs transduced
with the four factors were re-seeded at 0.5.times.10.sup.5 cells
per 100-mm dishes with feeder cells. Thirty to 40 days after
transduction, the colonies were selected for expansion.
[0254] Characterization of iPS cells. RT-PCR and teratoma formation
were performed as previously described. For the chimera
experiments, 15-20 iPS cells were injected into BDF1-derived
blastocysts, which were then transplanted into the uteri of
pseudo-pregnant mice.
Example 22
Establishment of Human iPS Cells from Epithelial Cells with Six
Factors
[0255] Among the nuclear reprogramming factors disclosed herein is
a nuclear reprogramming factor comprising one or more gene products
from the following genes: Klf4, c-Myc, Oct3/4, Sox2, Nanog, and
Lin28 (NCBI accession number NM.sub.--145833 (mouse) and
NM.sub.--024674 (human)). Establishment of induced pluripotent stem
cells was performed with combinations of these gene products. The
results are shown in TABLE 17.
TABLE-US-00017 TABLE 17 Summary of Experiments with Six Factors Day
23 Day 29 Day 23 non non non ES like ES like ES like ES like ES
like ES like 6F 59 39 167 42 16 27 -L 49 5 53 14 -N 220 11 216 47
-M 2 0 15 0 -O 0 0 0 0 -S 491 0 489 0 -K 61 0 51 0 -KS 1206 0 1305
0 -KO 0 0 0 0 -KM 0 0 0 0 0 0 -KN 51 0 57 0 -KL 28 0 41 0 -SO 0 0 0
0 -SM 0 0 0 0 -SN 188 0 171 0 -SL 112 0 136 0 -OM 0 0 0 0 -ON 0 0 0
0 -OL 0 0 0 0 -MN 3 0 8 0 -ML 0 0 0 0 -NL 98 1 119 9 17 6 GFP 0 0 0
0 KO 0 0 0 0 KS 0 0 0 0
[0256] 6.times.10.sup.6 293FT cells were plated on 10 cm dish and
cultured overnight, and then transfected with 3 .mu.g of
pLenti6/UbC-Slc7a1 lentiviral vector together with 9 .mu.g of
Virapower packaging mix by Lipofectamine 2000 (Invitrogen). After
24 hours, the culture medium was replaced with a fresh medium.
After 20 hours, the culture supernatant was collected and filtrated
through 0.45-.mu.m pore-size cellulose acetate filter (Whatman).
5.times.10.sup.5 epithelial cells were prepared on the previous
day. To the dish which the culture supernatant was removed from,
the aforementioned filtrated culture supernatant containing viruses
and 4 .mu.g/ml polybrene (Nacalai Tesque) were added. Then, the
cells were cultured for 24 hours.
[0257] In addition, 1.0.times.10.sup.6 Plat-E cells were plated on
6 cm dish and cultured. On the next day, the cells were transfected
with 9.0 .mu.g of pMX-based retrovirus vector incorporating klf4,
c-myc, oct3/4, sox2, nanog and/or Lin-28 by using 27 .mu.l of
Fugene6 transfection reagent. After 24 hours, the culture medium
was replaced with a fresh medium. On the next day, the culture
supernatant of Plat-E cells was collected and filtrated through
0.45-.mu.m pore-size cellulose acetate filter (Whatman). Seven days
after lentivirus infection, epithelial cells were plated at
3.0.times.10.sup.5 cells per 6 cm dish again, and the
aforementioned culture supernatant containing retrovirus and
polybrene were added thereto.
[0258] The term "6F" in TABLE 17 refers to the six factors (klf4,
c-myc, oct3/4, sox2, nanog and Lin-28), the term "L" refers to
Lin-28, the term "N" refers to nanog, the term "M" refers to c-Myc,
the term "O" refers to Oct3/4, the term "S" refers to Sox2, and the
term "K" refers to Klf4, respectively. The symbol "-" refers to
colonies obtained by subtracting from the six factors those factors
shown by the term subsequent to the symbol "-". For example, the
term "-L" shows the colonies obtained with the remaining five
factors other than lin-28, and the term "-KS" shows the colonies
obtained with the remaining four factors other than Klf4 and Sox2,
respectively.
[0259] The numbers in TABLE 17 refer to the number of colonies. The
term "non-ES like" refers to shows colonies having a non-ES cell
like morphology, and the term "ES like" refers to colonies having
an ES like cell morphology.
[0260] Two experimental results are shown. The first experiment
shows the number of colonies from cells introduced with various
combinations of factors 23 days or 29 days after gene introduction
and the second experiment shows the number of "6F," "-KM," and
"-NL." According to these experimental results, the number of
colonies from cells not-introduced with lin-28 such as "-L" was
larger than that of cell transduced with lin-28, suggesting that
Lin-28 plays an important role to improve the efficiency of
establishment of iPS cells.
[0261] In addition, iPS cell induction experiments were performed
with six factors (Klf4, c-Myc, Oct3/4, Sox2, Nanog, and Lin28) and
two different combinations of four factors (Klf4, c-Myc, Oct3/4 and
Sox2, referred to as Y4F in FIG. 49; and Oct3/4, Sox2, Nanog, and
Lin-28, referred to as T4F in FIG. 49). The second combination of
four factors, T4F, is the same combination as disclosed in Yu et
al., Science 318:1917-1920, 2007. In these experiments, use of the
six factors and the Y4F combination of four factors generated
colonies having a similar morphology as ES-like cell colony,
whereas the T4F combination generated no colonies having a similar
morphology as ES-like cell colony (FIG. 49).
Example 23
More Efficient iPS Cell Generation with Sall4
[0262] Experiments performed with mouse embryonic fibroblasts and
adult human dermal fibroblasts showed that iPS cell induction with
three factors (Klf4, Oct3/4, and Sox2) is more efficient when Sall4
is added to the combination, that is when Klf4, Oct3/4, Sox2, and
Sall4 are used (FIGS. 50(A)-(C) and FIG. 51). More ES like colonies
were also observed when Sall4 was added to the four factors (Klf4,
Oct3/4, Sox2, and c-Myc) or the three factors (Klf4, Oct3/4, Sox2)
under the experimental conditions used. These experiments show that
addition of Sall4 to the nuclear reprogramming factor can improve
iPS induction efficiency.
[0263] Kits of the Present Invention.
[0264] One aspect of the present invention includes kits designed
for use in the preparation and induction of iPS cells. Another
aspect of the invention comprises kits for the prevention or
treatment of a medical condition or disease through the use of an
NRF, an iPS cell or a cell derived from an iPS cell by induction of
differentiation. It is also an object of the present invention to
provide compositions and methods useful for in vitro transfection,
in vivo transfection, ex vivo transfection, in situ labeling,
diagnostic tests, genetic therapy, gene therapy, treatment of
medical conditions, and the creation of transgenic animals. One
aspect of the present invention comprises kits designed for in
vitro transfection, in vivo transfection, ex vivo transfection, in
situ labeling, diagnostic tests, genetic therapy, gene therapy,
treatment of medical conditions, and the creation of transgenic
animals. Accordingly, the present invention includes a composition
comprising one or more NRFs, one or more iPS cells, one or more
cells derived from an iPS cell or cells, and combinations
thereof.
UTILITY AND PRACTICAL APPLICATIONS
[0265] By using the nuclear reprogramming factor provided by the
present invention, reprogramming of differentiated cell nuclei can
be conveniently and highly reproducibly induced without using
embryos or ES cells, and induced pluripotent stem cells as
undifferentiated cells having differentiation ability, pluripotency
and growth ability similar to those of ES cells can be
established.
[0266] Uses of the induced pluripotent stem cells prepared by the
method of the present invention are not particularly limited. The
cells can be used for any experiments and research conducted with
ES cells, therapeutic treatments utilizing ES cells and the like.
For example, desired differentiated cells (e.g., nerve cells,
cardiac muscle cells, hemocyte cells and the like) can be derived
by treating induced pluripotent stem cells obtained by the method
of the present invention with retinoic acid, growth factors such as
EGF, glucocorticoid or the like, and stem cell therapy based on
cellular auto-transplantation can be achieved by returning the
differentiated cells obtained as described above to the patient.
However, uses of the induced pluripotent stem cells of the present
invention are not limited to the aforementioned specific
embodiments.
[0267] Thus, the present invention has enabled the generation of
iPS cells from adult human dermal fibroblasts and other human
somatic cells, which are indistinguishable from human ES cells in
their differentiation potential in vitro and in teratomas.
Furthermore, the instant invention allows for the generation of
patient- and disease-specific pluripotent stem cells. Even with the
presence of retroviral integration, human iPS cells are useful for
understanding disease mechanisms, drug screening, and toxicology.
For example, hepatocytes derived from iPS cells with various
genetic and disease backgrounds can be utilized in predicting liver
toxicity of drug candidates. Human iPS cells may overcome the
ethical issues that hES cells confront.
[0268] Reference is made to the following documents: Takahashi, et
al. Cell 131:861-872, 2007; Nakagawa et al. Nature Biotechnology
26(1):101-106, 2008; Takahashi et al., Cell 126: 663-676, 2006;
Okita et al., Nature 448:313-17, 2007; Takahashi, et al. Nature
Protocols 2(12):3081-89, 2007; and the supplementary figures and
data associated with these documents, all of which are incorporated
by reference herein in their entireties.
[0269] Although the present invention has been described in
considerable detail with regard to certain versions thereof, other
versions are possible, and alterations, permutations, and
equivalents of the versions shown will become apparent to those
skilled in the art upon a reading of the specification and study of
the drawings. Also, the various features of the versions herein can
be combined in various ways to provide additional versions of the
present invention. Furthermore, certain terminology has been used
for the purposes of descriptive clarity, and not to limit the
present invention. Therefore, any appended claims should not be
limited to the description of the preferred versions contained
herein and should include all such alterations, permutations, and
equivalents as fall within the true spirit and scope of the present
invention.
[0270] Having now fully described this invention, it will be
understood to those of ordinary skill in the art that the methods
of the present invention can be carried out with a wide and
equivalent range of conditions, formulations, and other parameters
without departing from the scope of the invention or any
embodiments thereof.
[0271] The attached Sequence Listing includes those sequences
disclosed in PCT/JP2006/324881, which is incorporated by reference
herein in its entirety.
Sequence CWU 1
1
133128DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1tgtggggccc tgaaaggcga gctgagat 28228DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2atgggccgcc atacgacgac gctcaact 28324DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
3gaagtctggt tccttggcag gatg 24420DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 4actcgataca ctggcctagc
20520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 5caggtgtttg agggtagctc 20620DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
6cggttcatca tggtacagtc 20724DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 7actgcccctc atcagactgc tact
24824DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 8cactgccttg tactcgggta gctg 24924DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
9gttccaacct gtgcctcgcg tctt 241026DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 10agcgaggcat ggagagagcg
gagcag 261123DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 11cgtggtgagc atcttcggag tgg
231223DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 12ccttcttggt ccgcccgttc tta 231330DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
13atggacgcaa ctgtgaacat gatgttcgca 301430DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
14ctttgaggtc ctggtccatc acgtgaccat 301524DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
15ccattagggg ccatcatcgc tttc 241624DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
16cactgctcac tggagggggc ttgc 241724DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
17tgctgcggtc caggccatca agag 241824DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
18gggcactgtt cagttcagcg gatc 241923DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
19tctttccacc aggcccccgg ctc 232023DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 20tgcgggcgga catggggaga tcc
232130DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 21attcttcgtt gtcaagccgc caaagtggag
302230DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 22agttgtttgc tgcggagttg tcatctcgtc
30232127DNASimian virus 40 23atggataaag ttttaaacag agaggaatct
ttgcagctaa tggaccttct aggtcttgaa 60aggagtgcct gggggaatat tcctctgatg
agaaaggcat atttaaaaaa atgcaaggag 120tttcatcctg ataaaggagg
agatgaagaa aaaatgaaga aaatgaatac tctgtacaag 180aaaatggaag
atggagtaaa atatgctcat caacctgact ttggaggctt ctgggatgca
240actgagattc caacctatgg aactgatgaa tgggagcagt ggtggaatgc
ctttaatgag 300gaaaacctgt tttgctcaga agaaatgcca tctagtgatg
atgaggctac tgctgactct 360caacattcta ctcctccaaa aaagaagaga
aaggtagaag accccaagga ctttccttca 420gaattgctaa gttttttgag
tcatgctgtg tttagtaata gaactcttgc ttgctttgct 480atttacacca
caaaggaaaa agctgcactg ctatacaaga aaattatgga aaaatattct
540gtaaccttta taagtaggca taacagttat aatcataaca tactgttttt
tcttactcca 600cacaggcata gagtgtctgc tattaataac tatgctcaaa
aattgtgtac ctttagcttt 660ttaatttgta aaggggttaa taaggaatat
ttgatgtata gtgccttgac tagagatcca 720ttttctgtta ttgaggaaag
tttgccaggt gggttaaagg agcatgattt taatccagaa 780gaagcagagg
aaactaaaca agtgtcctgg aagcttgtaa cagagtatgc aatggaaaca
840aaatgtgatg atgtgttgtt attgcttggg atgtacttgg aatttcagta
cagttttgaa 900atgtgtttaa aatgtattaa aaaagaacag cccagccact
ataagtacca tgaaaagcat 960tatgcaaatg ctgctatatt tgctgacagc
aaaaaccaaa aaaccatatg ccaacaggct 1020gttgatactg ttttagctaa
aaagcgggtt gatagcctac aattaactag agaacaaatg 1080ttaacaaaca
gatttaatga tcttttggat aggatggata taatgtttgg ttctacaggc
1140tctgctgaca tagaagaatg gatggctgga gttgcttggc tacactgttt
gttgcccaaa 1200atggattcag tggtgtatga ctttttaaaa tgcatggtgt
acaacattcc taaaaaaaga 1260tactggctgt ttaaaggacc aattgatagt
ggtaaaacta cattagcagc tgctttgctt 1320gaattatgtg gggggaaagc
tttaaatgtt aatttgccct tggacaggct gaactttgag 1380ctaggagtag
ctattgacca gtttttagta gtttttgagg atgtaaaggg cactggaggg
1440gagtccagag atttgccttc aggtcaggga attaataacc tggacaattt
aagggattat 1500ttggatggca gtgttaaggt aaacttagaa aagaaacacc
taaataaaag aactcaaata 1560tttccccctg gaatagtcac catgaatgag
tacagtgtgc ctaaaacact gcaggccaga 1620tttgtaaaac aaatagattt
taggcccaaa gattatttaa agcattgcct ggaacgcagt 1680gagtttttgt
tagaaaagag aataattcaa agtggcattg ctttgcttct tatgttaatt
1740tggtacagac ctgtggctga gtttgctcaa agtattcaga gcagaattgt
ggagtggaaa 1800gagagattgg acaaagagtt tagtttgtca gtgtatcaaa
aaatgaagtt taatgtggct 1860atgggaattg gagttttaga ttggctaaga
aacagtgatg atgatgatga agacagccag 1920gaaaatgctg ataaaaatga
agatggtggg gagaagaaca tggaagactc agggcatgaa 1980acaggcattg
attcacagtc ccaaggctca tttcaggccc ctcagtcctc acagtctgtt
2040catgatcata atcagccata ccacatttgt agaggtttta cttgctttaa
aaaacctccc 2100acacctcccc ctgaacctga aacataa 212724456DNAHuman
papilloma virus type 16 24atgtttcagg acccacagga gcgacccaga
aagttaccac agttatgcac agagctgcaa 60acaactatac atgatataat attagaatgt
gtgtactgca agcaacagtt actgcgacgt 120gaggtatatg actttgcttt
tcgggattta tgcatagtat atagagatgg gaatccatat 180gctgtatgtg
ataaatgttt aaagttttat tctaaaatta gtgagtatag acattattgt
240tatagtttgt atggaacaac attagaacag caatacaaca aaccgttgtg
tgatttgtta 300attaggtgta ttaactgtca aaagccactg tgtcctgaag
aaaagcaaag acatctggac 360aaaaagcaaa gattccataa tataaggggt
cggtggaccg gtcgatgtat gtcttgttgc 420agatcatcaa gaacacgtag
agaaacccag ctgtaa 45625297DNAHuman papilloma virus type 16
25atgcatggag atacacctac attgcatgaa tatatgttag atttgcaacc agagacaact
60gatctctact gttatgagca attaaatgac agctcagagg aggaggatga aatagatggt
120ccagctggac aagcagaacc ggacagagcc cattacaata ttgtaacctt
ttgttgcaag 180tgtgactcta cgcttcggtt gtgcgtacaa agcacacacg
tagacattcg tactttggaa 240gacctgttaa tgggcacact aggaattgtg
tgccccatct gttctcagaa accataa 2972623DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
26ccccagggcc ccattttggt acc 232726DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 27ggcacccctg gcatggctct
tggctc 262825DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 28acgatcgtgg ccccggaaaa ggacc
252927DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 29caacaaccga aaatgcacca gccccag
273024DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 30ttatcgtcga ccactgtgct gctg 243124DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
31ccctttttct ggagactaaa taaa 243224DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
32gacaggggga ggggaggagc tagg 243326DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
33cttccctcca accagttgcc ccaaac 263426DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
34gggaaatggg aggggtgcaa aagagg 263526DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
35ttgcgtgagt gtggatggga ttggtg 263625DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
36cagccccgat tcttccacca gtccc 253725DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
37cggaagattc ccagtcgggt tcacc 253824DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
38cttatgctac gtaaaggagc tggg 243924DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
39gtgccaaccc aggtcccgga agtt 244024DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
40cagatcctaa acagctcgca gaat 244123DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
41gcgtacgcaa attaaagtcc aga 234223DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 42ctacaacgcc tacgagtcct aca
234324DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 43gttgcaccag aaaagtcaga gttg 244424DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
44atatcccgcc gtgggtgaaa gttc 244524DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
45actcagccat ggactggagc atcc 244624DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
46ggagccgcct gccctggaaa attc 244724DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
47tttttcctga tattctattc ccat 244824DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
48ccgtccccgc aatctccttc catc 244924DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
49atgatgccaa catggctccc ggtg 245026DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
50cctgctcaag ctgactcgac accgtg 265125DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
51ggaaaagctg gccctggggt ggagc 255228DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
52tgattgtagt gctttctggc tgggctcc 285326DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
53gcgtcctggg aagggagatc cggagc 265426DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
54ttgaggggca tcgtcgcggg aggctg 265524DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
55cgagaggacc ccgtggatgc agag 245624DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
56ggcggccatc ttcagcttct ccag 245726DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
57gccctctccc tcccctccac gcacag 265826DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
58cggcgccgtt gctcacagac cacagg 265926DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
59ggcccgccac ttgcaggagt accagg 266026DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
60cttctgctcg ggcccctcat gagacg 266128DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
61acccattatc cagatgtgtt tgcccgag 286226DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
62atggtgaagc tgggcatagg cggcag 266326DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
63tgggagcggt gaagatggaa gggcac 266426DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
64tcatgccagc gcccacgtac gacgac 266526DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
65cgctttcatg gtgtgggcta aggacg 266626DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
66tagttggggt ggtcctgcat gtgctg 266726DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
67cgccaggatc cccgctttga aatctg 266826DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
68tcggccgcca gctctttgat gtgttc 266924DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
69ggaggcgtgg agctcagcga cacc 247024DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
70cggggagctc gctgacggag tctg 247130DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
71caggtggcgg acgtgtgaaa attgagagtg 307226DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
72cacgctggat ctgcctgggg actgtg 267326DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
73acagagggga ggtgcgccag ttcacg 267426DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
74acggggtgga cctcgctgca cagatc 267526DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
75ggcaccagca gcagcaggag cagcag 267626DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
76ccacgtctga ggagccgagg aagcag 267726DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
77gggccccatc aacttcaccg tcttcc 267826DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
78tgtagtcgat gttccccgcc aggtcc 267926DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
79atgagcggga gaaggagcgg cagaac 268026DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
80tcaatggcca gcaccttcct cctctc 268128DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
81tttaacaccg ccagcgctct tcaccttg 288228DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
82tcgtggcgcg tgtgttgtgg gtatctcg 288325DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
83ctggaggccg agcagaagcg caacg 258425DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
84gtccgcccgc tcctctgcct catcc 258520DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
85tttttttttt tttttttttt 208627DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 86gccacagcaa acctcctcac
agcccac 278728DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 87ctcgtcgttt ccgcaacaag tcctcttc
288826DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 88caccatggcg ggacacctgg cttcag 268926DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
89acctcagttt gaatgcatgg gagagc 269031DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
90caccatgtac aacatgatgg agacggagct g 319126DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
91tcacatgtgt gagaggggca gtgtgc 269230DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
92caccatggct gtcagtgacg cgctgctccc
309330DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 93ttaaaaatgt ctcttcatgt gtaaggcgag
309430DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 94caccatgccc ctcaacgtta gcttcaccaa
309530DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 95tcacgcacaa gagttccgta gctgttcaag
309627DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 96caccatgggc tgcaaaaacc tgctcgg
279727DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 97tcatttgcac tggtccaagt tgctgtc
279833DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 98attgtcgacg gggatttggc agggtcacag gac
339932DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 99cccagatctc caatgccacc tcctcccaaa cg
3210032DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 100cactcgaggt ggaggagctg agggcactgt gg
3210132DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 101cacagatctg aaatgagggc ttgcgaaggg ac
3210230DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 102ggtttaaaag ggtaaatgtg attatattta
3010322DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 103caaactacaa ccacccatca ac 2210430DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
104gaggttggag tagaaggatt gttttggttt 3010530DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
105cccccctaac ccatcacctc caccacctaa 3010625DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
106tggttaggtt ggttttaaat ttttg 2510726DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
107aacccaccct tataaattct caatta 2610833DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
108caccatgtac agcatgatga tggagaccga cct 3310926DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
109ctagatatgc gtcaggggca ccgtgc 2611034DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
110caccatgtac agcctgctgg agactgaact caag 3411130DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
111tcagatgtgg gtcagcggca ccgttccatt 3011227DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
112cacctcggcc atggcctcgc tgctggg 2711327DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
113ctccattcct ccagctctat gacacac 2711428DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
114caccatggcg ctgaccagct cctcacaa 2811524DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
115ttaaaggtgg gttactggca tggg 2411626DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
116caccagagcc atgagcagcc cggatg 2611726DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
117cgtcaaatgt cggggtagtt gcaata 2611829DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
118caccatgcag agatcgccgc ccggctacg 2911930DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
119ctagcctgag atgcaagcac tgtaatagac 3012028DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
120caccatgaat aatccatcag aaaccaat 2812124DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
121gctctgcact cagctcactg tgcc 2412228DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
122caccatggcc accaccgcgc agtatctg 2812324DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
123ggaacccagt ccgcagggtc actg 2412429DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
124caccatgagg cagaagagag agaggaggc 2912530DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
125tcagaggtga cgcttcatgt gcagagctaa 3012630DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
126caccatggcg ctcagcgagc ctatcttgcc 3012730DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
127ctacatatgt cgcttcatgt gcaaggccag 3012828DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
128caccatgccc acgcgggtgc tgaccatg 2812924DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
129tcgctcagtt ctggtggcgc ttca 2413034DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
130caccatggac ttcgactcgt atcagcacta tttc 3413130DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
131ttagtagcca ctgaggtacg cgattctctt 3013230DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
132caccatgccc agctgcaccg cgtccaccat 3013326DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
133ttagcaagtc cgagcgtgtt cgatct 26
* * * * *
References