U.S. patent application number 14/414210 was filed with the patent office on 2015-07-02 for novel cardiomyocyte marker.
The applicant listed for this patent is KYOTO UNIVERSITY. Invention is credited to Shunsuke Funakoshi, Shinya Yamanaka, Yoshinori Yoshida.
Application Number | 20150184129 14/414210 |
Document ID | / |
Family ID | 49948939 |
Filed Date | 2015-07-02 |
United States Patent
Application |
20150184129 |
Kind Code |
A1 |
Yamanaka; Shinya ; et
al. |
July 2, 2015 |
NOVEL CARDIOMYOCYTE MARKER
Abstract
The present invention provides a method for producing or
detecting cardiomyocytes, which method comprises
extracting/detecting cardiomyocytes from a cell population
comprising cardiomyocytes using, as an index, positivity of NCAM1,
SSEA3, SSEA4 and/or CD340.
Inventors: |
Yamanaka; Shinya; (Kyoto,
JP) ; Yoshida; Yoshinori; (Kyoto, JP) ;
Funakoshi; Shunsuke; (Kyoto, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KYOTO UNIVERSITY |
Kyoto |
|
JP |
|
|
Family ID: |
49948939 |
Appl. No.: |
14/414210 |
Filed: |
July 16, 2013 |
PCT Filed: |
July 16, 2013 |
PCT NO: |
PCT/JP2013/069721 |
371 Date: |
January 12, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61672637 |
Jul 17, 2012 |
|
|
|
Current U.S.
Class: |
435/7.21 ;
435/34; 530/389.1 |
Current CPC
Class: |
C12N 2500/02 20130101;
C12N 2506/45 20130101; C12N 5/0657 20130101; C12N 2501/415
20130101; G01N 2333/91205 20130101; C12N 2501/115 20130101; G01N
2405/10 20130101; G01N 33/74 20130101; G01N 33/566 20130101; C12N
2501/155 20130101; G01N 33/56966 20130101; G01N 33/5005 20130101;
G01N 33/92 20130101; C12N 2501/165 20130101; C12N 2501/20
20130101 |
International
Class: |
C12N 5/077 20060101
C12N005/077; G01N 33/92 20060101 G01N033/92; G01N 33/569 20060101
G01N033/569 |
Claims
1. A method for producing a cardiomyocyte(s), comprising a)
providing a cell population comprising the cardiomyocyte(s), and b)
extracting a cardiomyocyte(s) from the cell population comprising
the cardiomyocyte(s) using, as an index, positivity of at least one
selected from the group consisting of NCAM1, SSEA3, SSEA4 and
CD340.
2. The method according to claim 1, wherein said cardiomyocyte is
human cardiomyocyte.
3. The method according to claim 1, wherein said cell population
comprising cardiomyocyte(s) is a cell population obtained by
inducing differentiation of a pluripotent stem cell(s), or a cell
population composed of cells of an isolated tissue(s).
4. The method according to claim 3, wherein said pluripotent stem
cell is an ES cell or iPS cell.
5. The method according to claim 3, wherein said induction of
differentiation into a cardiomyocyte(s) comprises forming an
embryoid body.
6. The method according to claim 4, wherein said induction of
differentiation into a cardiomyocyte(s) comprises culturing an
embryoid body in a medium comprising a cytokine(s).
7. The method according to claim 6, wherein said cytokine(s) is at
least one cytokine selected from the group consisting of activin A,
BMP4, b-FGF, and VEGF.
8. The method according to claim 6, wherein said medium further
comprises a Wnt inhibitor.
9. The method according to claim 8, wherein said Wnt inhibitor is
DKK-1.
10. A method for detecting a cardiomyocyte(s), comprising detecting
a cardiomyocyte(s) in a cell population comprising a cardiomyocyte
using, as an index, positivity of at least one selected from the
group consisting of NCAM1, SSEA3, SSEA4 and CD340.
11. The method according to claim 10, wherein said cardiomyocyte is
human cardiomyocyte.
12. The method according to claim 10, wherein said cell population
comprising a cardiomyocyte is a cell population obtained by
inducing differentiation of a pluripotent stem cell(s), or a cell
population composed of cells of an isolated tissue(s).
13. The method according to claim 12, wherein said pluripotent stem
cell is an ES cell or iPS cell.
14. A kit for extracting or detecting a cardiomyocyte(s),
comprising a reagent for detecting at least one selected from the
group consisting of NCAM1, SSEA3, SSEA4 and CD340.
15. The kit according to claim 14, wherein said cardiomyocyte is a
human cardiomyocyte.
Description
FIELD OF INVENTION
[0001] The present invention relates to novel cardiomyocyte markers
for extracting/detecting cardiomyocytes from a cell population
comprising the cardiomyocytes.
BACKGROUND ART
[0002] Since cardiomyocytes lose their division potential at the
time of birth and hence their regeneration is difficult, recent
interest has focused on replacement therapy wherein cardiomyocytes
obtained by inducing differentiation of cells having pluripotency
(WO/2007/069666), such as embryonic stem cells (ES cells) or
induced pluripotent stem cells (iPS cells), are transplanted to a
cardiac tissue damaged due to myocardial infarction, myocarditis,
aging or the like. Although many methods for inducing
differentiation of such pluripotent stem cells into cardiomyocytes
have been reported (WO2007/002136, WO2009/118928 and Yan P, et al.,
Biochem Biophys Res Commun. 379:115-20 (2009)), use of the induced
cells in transplantation further requires enhancement of the purity
of the cardiomyocytes by sorting or the like. Although, at present,
CD 166 (ALCM) (Rust W, et al., Regen Med. 4, 225-37 (2009)),
N-cadherin (JP 2010-158206 A and Honda M, et al., Biochem Biophys
Res Commun. 29, 351, 877-82 (2006)), VCAM1 (International
Application No. PCT/JP2012/059617) and the like have been reported
as surface markers for cardiomyocytes, it is thought that more
markers are necessary for enhancement of the purity of
cardiomyocytes.
SUMMARY OF THE INVENTION
[0003] An object of the present invention is to extract/detect
cardiomyocytes from a cell population comprising the
cardiomyocytes. Accordingly, the present invention aims to provide
markers specific for cardiomyocytes.
[0004] In order to solve the above problem, the inventors of the
present invention focused on NCAM1, SSEA3, SSEA4 and CD340, and
discovered that, by using positivity of at least one of these as an
index, cardiomyocytes can be obtained at high efficiency from a
cell population containing cardiomyocytes obtained by inducing
differentiation of pluripotent stem cells.
[0005] Based on the above knowledge, the inventors of the present
invention succeeded in isolation and purification of cardiomyocytes
by using, as an index, positivity of at least one selected from the
group consisting of NCAM1, SSEA3, SSEA4 and CD340, thereby
completed the present invention.
[0006] It is one aspect of the present invention to provide a
method for producing a cardiomyocyte(s), comprising
a) providing a cell population comprising the cardiomyocyte(s), and
b) extracting a cardiomyocyte(s) from the cell population
comprising the cardiomyocyte(s) using, as an index, positivity of
at least one selected from the group consisting of NCAM1, SSEA3,
SSEA4 and CD340.
[0007] It is another aspect of the present invention to provide the
method as described above, wherein said cardiomyocyte is human
cardiomyocyte.
[0008] It is another aspect of the present invention to provide the
method as described above, wherein said cell population comprising
cardiomyocyte(s) is a cell population obtained by inducing
differentiation of a pluripotent stem cell(s), or a cell population
composed of cells of an isolated tissue(s).
[0009] It is another aspect of the present invention to provide the
method as described above, wherein said pluripotent stem cell is an
ES cell or iPS cell.
[0010] It is another aspect of the present invention to provide the
method as described above, wherein said induction of
differentiation into a cardiomyocyte(s) comprises forming an
embryoid body.
[0011] It is another aspect of the present invention to provide the
method as described above, wherein said induction of
differentiation into a cardiomyocyte(s) comprises culturing an
embryoid body in a medium comprising a cytokine(s).
[0012] It is another aspect of the present invention to provide the
method as described above, wherein said cytokine(s) is at least one
cytokine selected from the group consisting of activin A, BMP4,
b-FGF, and VEGF.
[0013] It is another aspect of the present invention to provide the
method as described above, wherein said medium further comprises a
Wnt inhibitor.
[0014] It is another aspect of the present invention to provide the
method as described above, wherein said Wnt inhibitor is DKK-1.
[0015] It is another aspect of the present invention to provide a
method for detecting a cardiomyocyte(s), comprising detecting a
cardiomyocyte(s) in a cell population comprising a cardiomyocyte
using, as an index, positivity of at least one selected from the
group consisting of NCAM1, SSEA3, SSEA4 and CD340.
[0016] It is another aspect of the present invention to provide the
method as described above, wherein said cardiomyocyte is human
cardiomyocyte.
[0017] It is another aspect of the present invention to provide the
method as described above, wherein said cell population comprising
a cardiomyocyte is a cell population obtained by inducing
differentiation of a pluripotent stem cell(s), or a cell population
composed of cells of an isolated tissue(s).
[0018] It is another aspect of the present invention to provide the
method as described above, wherein said pluripotent stem cell is an
ES cell or iPS cell.
[0019] It is another aspect of the present invention to provide a
kit for extracting or detecting a cardiomyocyte(s), comprising a
reagent for detecting at least one selected from the group
consisting of NCAM1, SSEA3, SSEA4 and CD340.
[0020] It is another aspect of the present invention to provide the
kit as described above, wherein said cardiomyocyte is a human
cardiomyocyte.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is a protocol for inducing differentiation of
pluripotent cells into cardiomyocytes.
[0022] FIG. 2 illustrates the results of flow cytometry on Day 0,
Day 4, Day 8, Day 12, Day 16, Day 20, Day 26, Day 33, Day 45 and
Day 60 after the beginning of induction of differentiation of the
201B7-M6GIP4 strain. In the figure, the ordinate indicates the
intensity of NCAM1, and the abscissa indicates the intensity of
EGFP.
[0023] FIG. 3 illustrates the results of flow cytometry on Day 30
after the beginning of induction of differentiation of the 606A1
strain. In the left column, the abscissa indicates the intensity of
NCAM1, and, in the right column, the abscissa indicates the
intensity of Troponin T.
[0024] FIG. 4 illustrates the results of flow cytometry on Day 0,
Day 4, Day 8, Day 12, Day 16, Day 20, Day 26, Day 33, Day 45 and
Day 60 after the beginning of induction of differentiation of the
201B7-M6GIP4 strain. In the figure, the ordinate indicates the
intensity of SSEA3, and the abscissa indicates the intensity of
EGFP.
[0025] FIG. 5 illustrates the results of flow cytometry on Day 0,
Day 4, Day 8, Day 12, Day 16, Day 20, Day 26, Day 33, Day 45 and
Day 60 after the beginning of induction of differentiation of the
201B7-M6GIP4 strain. In the figure, the ordinate indicates the
intensity of SSEA4, and the abscissa indicates the intensity of
EGFP.
[0026] FIG. 6 illustrates the results of flow cytometry on Day 0,
Day 4, Day 8, Day 12, Day 16, Day 20, Day 26, Day 33 and Day 45
after the beginning of induction of differentiation of the 201
B7-M6GIP4 strain. In the figure, the ordinate indicates the
intensity of CD340, and the abscissa indicates the intensity of
EGFP.
EMBODIMENTS FOR CARRYING OUT THE INVENTION
[0027] The present invention is described below in detail.
[0028] As described below, the present invention relates to a
method for producing a cardiomyocyte(s), comprising extracting a
cardiomyocyte(s) from a cell population comprising a cardiomyocyte
using, as an index, positivity of at least one selected from the
group consisting of NCAM1, SSEA3, SSEA4 and CD340, and a method for
detecting a cardiomyocyte(s), comprising detecting a
cardiomyocyte(s) in a cell population comprising a cardiomyocyte
using, as an index, positivity of at least one selected from the
group consisting of NCAM1, SSEA3, SSEA4 and CD340.
[0029] The origin of the cell population comprising a cardiomyocyte
is not restricted as long as the cell population comprises a
cardiomyocyte. Examples of the cell population include cells
contained in peripheral blood, heart, myeloid tissue, adipose
tissue, skeletal muscle tissue, amniotic tissue, placental tissue,
umbilical cord blood or the like obtained by an arbitrary method,
and cells obtained by inducing differentiation of pluripotent stem
cells.
[0030] The term "extraction of cardiomyocytes" means that the ratio
of cardiomyocytes is increased relative to other types of cells,
and preferably means that the cardiomyocytes are concentrated to a
ratio of not less than 50%, 60%, 70%, 80% or 90%. The term more
preferably means that a cell population comprising cardiomyocytes
at a ratio of 100% is obtained.
[0031] NCAM1 is the gene known as "neural cell adhesion molecule
1", CD56, NCAM or MSK39, and is involved in cell adhesion. For
example, in the case of human, NCAM1 is the gene described in NCBI
(National Center for Biotechnology Information) accession No.
NM.sub.--000615, NM.sub.--001076682, NM.sub.--001242607 or
NM.sub.--181351 or the protein encoded by thereby. NCAM1 also
includes isoforms produced by alternative splicing.
[0032] SSEA3 and SSEA4 are epitopes on related glycosphingolipids
(GSLs), termed GL-5 and GL-70, which are known as
Anti-Stage-Specific Embryonic Antigen-3 and Anti-Stage-Specific
Embryonic Antigen-4, respectively. Also, SSEA3 and SSEA4 are known
to be recognized by MC631 antibody and MC813-70 antibody,
respectively (Brimble SN et al., Stem Cells. 2007 Jan;
25(1):54-62.). Since SSEA3 and SSEA4 are specifically expressed on
the surface of stem cells, these are generally used as stem cell
markers.
[0033] CD340 is the member of the EGF receptor family known as
"v-erb-b2 erythroblastic leukemia viral oncogene homolog 2"
(ERBB2), HER-2/neu or the like. For example, in the case of human,
CD340 is the gene described in NCBI (National Center for
Biotechnology Information) accession No. NM.sub.--001005862 or
NM.sub.--004448 or the protein encoded by thereby.
[0034] When cardiomyocytes are extracted from a cell population
comprising cardiomyocytes using as an index NCAM1, SSEA3, SSEA4
and/or CD340, each of these genes or the proteins encoded thereby
may be used alone, or two or more of these may be used in an
arbitrary combination. In cases where NCAM1, SSEA3, SSEA4 and CD340
are used in combination, the concentration ratio of cardiomyocytes
can be preferably increased compared to cases where each of these
is used alone.
[0035] The term "cardiomyocyte" means a cell of a cardiac muscle
having the property of self-beating. The cardiomyocyte can be
characterized by being positive for cardiac troponin (cTnT or
troponin T type 2) and/or aMHC (a myosin heavy chain), which are
myocardial markers.
<Pluripotent Stem Cells>
[0036] The pluripotent stem cells which may be used in the present
invention are stem cells having pluripotency which enables the
cells to differentiate into any cells existing in the living body,
which stem cells also have growth ability. Examples of the
pluripotent stem cells include, but are not limited to, embryonic
stem (ES) cells, embryonic stem cells derived from a cloned embryo
obtained by nuclear transfer (ntES cells), germline stem cells ("GS
cells"), embryonic germ cells ("EG cells") and induced pluripotent
stem (iPS) cells. The pluripotent stem cells are preferably ES
cells, ntES cells or iPS cells.
(A) Embryonic Stem Cells
[0037] ES cells are stem cells established from the inner cell mass
of an early embryo (for example, blastocyst) of a mammal such as
human or mouse, which cells have pluripotency and growth ability by
self-renewal.
[0038] ES cells are embryo-derived stem cells originated from the
inner cell mass of a blastocyst which is the embryo formed
following the 8-cell stage and the morula stage of a fertilized
egg, and ES cells have ability to differentiate into any cells
constituting an adult, that is, the so called pluripotency of
differentiation, and growth ability by self-renewal. ES cells were
discovered in mouse in 1981 (M. J. Evans and M. H. Kaufman (1981),
Nature 292:154-156), and this was followed by establishment of ES
cell lines of primates such as human and monkey (J. A. Thomson et
al. (1998), Science 282:1145-1147; J. A. Thomson et al. (1995),
Proc. Natl. Acad. Sci. USA, 92:7844-7848; J. A. Thomson et al.
(1996), Biol. Reprod., 55:254-259; J. A. Thomson and V. S. Marshall
(1998), Curr. Top. Dev. Biol., 38:133-165).
[0039] ES cells can be established by removing the inner cell mass
from the blastocyst of a fertilized egg of the subject animal,
followed by culturing the inner cell mass on fibroblasts as
feeders. The cells can be maintained by subculturing using a medium
supplemented with substances such as leukemia inhibitory factor
(LIF) and/or basic fibroblast growth factor (bFGF). Methods of
establishment and maintenance of human and monkey ES cells are
described in, for example, U.S. Pat. No. 5,843,780 B; Thomson JA,
et al. (1995), Proc Natl. Acad. Sci. U S A. 92:7844-7848; Thomson
JA, et al. (1998), Science. 282:1145-1147; H. Suemori et al.
(2006), Biochem. Biophys. Res. Commun., 345:926-932; M. Ueno et al.
(2006), Proc. Natl. Acad. Sci. USA, 103:9554-9559; H. Suemori et
al. (2001), Dev. Dyn., 222:273-279; H. Kawasaki et al. (2002),
Proc. Natl. Acad. Sci. USA, 99:1580-1585; and Klimanskaya I, et al.
(2006), Nature. 444:481-485.
[0040] In terms of the medium for preparation of ES cells, human ES
cells can be maintained, for example, using DMEM/F-12 medium
supplemented with 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential
amino acids, 2 mM L-glutamic acid, 20% KSR and 4 ng/ml bFGF at
37.degree. C. under a moist atmosphere of 2% CO.sub.2/98% air (O.
Fumitaka et al. (2008), Nat. Biotechnol., 26:215-224). Further, ES
cells need to be subcultured every 3 to 4 days, and the subculture
can be carried out using 0.25% trypsin and 0.1 mg/ml collagenase IV
in PBS supplemented with 1 mM CaCl.sub.2 and 20% KSR.
[0041] Selection of ES cells can be generally carried out by the
Real-Time PCR method using as an index/indices expression of a gene
marker(s) such as alkaline phosphatase, Oct-3/4 and/or Nanog. In
particular, for selection of human ES cells, expression of a gene
marker(s) such as OCT-3/4, NANOG and/or ECAD can be used as an
index/indices (E. Kroon et al. (2008), Nat. Biotechnol.,
26:443-452).
[0042] For example, in terms of human ES cell lines, WA01(H1) and
WA09(H9) can be obtained from WiCell Research Institute, and
KhES-1, KhES-2 and KhES-3 can be obtained from Institute for
Frontier Medical Sciences, Kyoto University (Kyoto, Japan).
(B) Germline Stem Cells
[0043] Germline stem cells are pluripotent stem cells derived from
testis, and play a role as the origin for spermatogenesis.
Similarly to ES cells, these cells can be induced to differentiate
into various series of cells, and, for example, have a property to
enable preparation of a chimeric mouse by transplantation of the
cells to a mouse blastocyst (M. Kanatsu-Shinohara et al. (2003)
Biol. Reprod., 69:612-616; K. Shinohara et al. (2004), Cell,
119:1001-1012). Germline stem cells are capable of self-renewal in
a medium containing glial cell line-derived neurotrophic factor
(GDNF), and, by repeating subculture under the same culture
conditions as those for ES cells, germline stem cells can be
obtained (Masanori Takehashi et al. (2008), Experimental Medicine,
26(5) (extra edition):41-46, Yodosha (Tokyo, Japan)).
(C) Embryonic Germ Cells
[0044] Embryonic germ cells are established from fetal primordial
germ cells and have pluripotency similar to that of ES cells. They
can be established by culturing primordial germ cells in the
presence of substances such as LIF, bFGF and stem cell factor (Y.
Matsui et al. (1992), Cell, 70:841-847; J. L. Resnick et al.
(1992), Nature, 359:550-551).
(D) Induced Pluripotent Stem Cells
[0045] Induced pluripotent stem (iPS) cells can be prepared by
introducing specific reprogramming factors to somatic cells, which
reprogramming factors are in the form of DNA or protein. iPS cells
are somatic cell-derived artificial stem cells having properties
almost equivalent to those of ES cells, such as pluripotency of
differentiation and growth ability by self-renewal (K. Takahashi
and S. Yamanaka (2006) Cell, 126:663-676; K. Takahashi et al.
(2007), Cell, 131:861-872; J. Yu et al. (2007), Science,
318:1917-1920; Nakagawa, M. et al., Nat. Biotechnol. 26:101-106
(2008); WO 2007/069666). The reprogramming factors may be
constituted by genes or gene products thereof, or non-coding RNAs,
which are expressed specifically in ES cells; or genes or gene
products thereof, non-coding RNAs or low molecular weight
compounds, which play important roles in maintenance of the
undifferentiated state of ES cells. Examples of the genes included
in the reprogramming factors include Oct3/4, Sox2, Sox1, Sox3,
Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15,
ERas, ECAT15-2, Tc11, beta-catenin, Lin28b, Sal11, Sa114, Esrrb,
Nr5a2 and Tbx3, and these reprogramming factors may be used either
individually or in combination. Examples of the combination of the
reprogramming factors include those described in WO2007/069666;
WO2008/118820; WO2009/007852; WO2009/032194; WO2009/058413;
WO2009/057831; WO2009/075119; WO2009/079007; WO2009/091659;
WO2009/101084; WO2009/101407; WO2009/102983; WO2009/114949;
WO2009/117439; WO2009/126250; WO2009/126251; WO2009/126655;
WO2009/157593; WO2010/009015; WO2010/033906; WO2010/033920;
WO2010/042800; WO2010/050626; WO 2010/056831; WO2010/068955;
WO2010/098419; WO2010/102267; WO 2010/111409; WO 2010/111422;
WO2010/115050; WO2010/124290; WO2010/147395; WO2010/147612; Huangfu
D, et al. (2008), Nat. Biotechnol., 26: 795-797; Shi Y, et al.
(2008), Cell Stem Cell, 2: 525-528; Eminli S, et al. (2008), Stem
Cells. 26:2467-2474; Huangfu D, et al. (2008), Nat Biotechnol.
26:1269-1275; Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574;
Zhao Y, et al. (2008), Cell Stem Cell, 3:475-479; Marson A, (2008),
Cell Stem Cell, 3, 132-135; Feng B, et al. (2009), Nat Cell Biol.
11:197-203; R. L. Judson et al., (2009), Nat. Biotech., 27:459-461;
Lyssiotis C A, et al. (2009), Proc Natl Acad Sci U S A.
106:8912-8917; Kim J B, et al. (2009), Nature. 461:649-643; Ichida
J K, et al. (2009), Cell Stem Cell. 5:491-503; Heng J C, et al.
(2010), Cell Stem Cell. 6:167-74; Han J, et al. (2010), Nature.
463:1096-100; and Mali P, et al. (2010), Stem Cells.
28:713-720.
[0046] Examples of the above-described reprogramming factors also
include histone deacetylase (HDAC) inhibitors [for example, low
molecular weight inhibitors such as valproic acid (VPA),
trichostatin A, sodium butyrate, MC 1293 and M344; and nucleic
acid-type expression inhibitors such as siRNAs and shRNAs against
HDAC (e.g., HDAC1 siRNA Smartpool.RTM. (Millipore) and HuSH 29mer
shRNA Constructs against HDAC1 (OriGene))], MEK inhibitors (for
example, PD 184352, PD98059, U0126, SL327 and PD0325901), Glycogen
synthase kinase-3 inhibitors (for example, Bio and CHIR99021), DNA
methyltransferase inhibitors (for example, 5'-azacytidine), histone
methyltransferase inhibitors (for example, low molecular weight
inhibitors such as BIX-01294; and nucleic acid-type expression
inhibitors such as siRNAs and shRNAs against Suv39h1, Suv39h2,
SetDB1 and G9a), L-channel calcium agonists (for example,
Bayk8644), butyric acid, TGF.beta. inhibitors or ALK5 inhibitors
(for example, LY364947, SB431542, 616453 and A-83-01), p53
inhibitors (for example, siRNAs and shRNAs against p53), ARID3A
inhibitors (for example, siRNAs and shRNAs against ARID3A), miRNAs
such as miR-291-3p, miR-294, miR-295 and mir-302, Wnt Signaling
(for example, soluble Wnt3a), neuropeptide Y, prostaglandins (for
example, prostaglandin E2 and prostaglandin J2), hTERT, SV4OLT,
UTF1, IRX6, GLIS1, PITX2 and DMRTB1, which are employed for
enhancing the establishment efficiency, and, in the present
description, these factors employed for the purpose of enhancement
of the establishment efficiency are not particularly distinguished
from reprogramming factors.
[0047] In cases where the reprogramming factors are in the form of
protein, the reprogramming factors may be introduced into somatic
cells by a method such as lipofection, fusion with a cell-permeable
peptide (e.g., HIV-derived TAT or polyarginine), or
microinjection.
[0048] In cases where the reprogramming factors are in the form of
DNA, the reprogramming factors may be introduced into somatic cells
by a method such as use of a vector including virus, plasmid and
artificial chromosome vectors; lipofection; use of liposome; or
microinjection. Examples of the virus vector include retrovirus
vectors, lentivirus vectors (these are described in Cell, 126, pp.
663-676, 2006; Cell, 131, pp. 861-872, 2007; and Science, 318, pp.
1917-1920, 2007), adenovirus vectors (Science, 322, 945-949, 2008),
adeno-associated virus vectors and Sendai virus vectors (WO
2010/008054). Examples of the artificial chromosome vector include
human artificial chromosome (HAC), yeast artificial chromosome
(YAC), and bacterial artificial chromosome (BAC and PAC). Examples
of the plasmid which may be used include plasmids for mammalian
cells (Science, 322:949-953, 2008). The vector may contain a
regulatory sequence(s) such as a promoter, enhancer, ribosome
binding sequence, terminator and/or polyadenylation site to enable
expression of the nuclear reprogramming factors; and, as required,
a sequence of a selection marker such as a drug resistance gene
(e.g., kanamycin-resistant gene, ampicillin-resistant gene or
puromycin-resistant gene), thymidine kinase gene or diphtheria
toxin gene; a gene sequence of a reporter such as the
green-fluorescent protein (GFP), .beta.-glucuronidase (GUS) or
FLAG; and/or the like. Further, in order to remove, after
introduction of the above vector into somatic cells, the genes
encoding the reprogramming factors, or both the promoter(s) and the
genes encoding the reprogramming factors linked thereto, the vector
may have LoxP sequences upstream and downstream of these
sequences.
[0049] Further, in cases where the reprogramming factors are in the
form of RNA, each reprogramming factor may be introduced into
somatic cells by a method such as lipofection or microinjection,
and an RNA into which 5-methylcytidine and pseudouridine (TriLink
Biotechnologies) were incorporated may be used in order to suppress
degradation (Warren L, (2010) Cell Stem Cell. 7:618-630).
[0050] Examples of the medium for induction of the iPS cells
include the DMEM, DMEM/F12 and DME media supplemented with 10 to
15% FBS (these media may further contain LIF,
penicillin/streptomycin, puromycin, L-glutamine, non-essential
amino acids, .beta.-mercaptoethanol and/or the like, as
appropriate); and commercially available media [for example, medium
for culturing mouse ES cells (TX-WES medium, Thromb-X), medium for
culturing primate ES cells (medium for primate ES/iPS cells,
ReproCELL) and serum-free medium (mTeSR, Stemcell Technology)].
[0051] Examples of the culture method include a method wherein
somatic cells and reprogramming factors are brought into contact
with each other at 37.degree. C. in the presence of 5% CO.sub.2 on
DMEM or DMEM/F12 medium supplemented with 10% FBS, and the cells
are cultured for about 4 to 7 days, followed by plating the cells
on feeder cells (e.g., mitomycin C-treated STO cells or SNL cells)
and starting culture in a bFGF-containing medium for culturing
primate ES cells about 10 days after the contact between the
somatic cells and the reprogramming factors, thereby allowing
iPS-like colonies to appear about 30 to about 45 days after the
contact, or later.
[0052] Alternatively, the cells may be cultured at 37.degree. C. in
the presence of 5% CO.sub.2 on feeder cells (e.g., mitomycin
C-treated STO cells or SNL cells) in DMEM medium supplemented with
10% FBS (this medium may further contain LIF,
penicillin/streptomycin, puromycin, L-glutamine, non-essential
amino acids, (3-mercaptoethanol and/or the like, as appropriate)
for about 25 to about 30 days or longer, thereby allowing ES-like
colonies to appear. Preferred examples of the culture method
include a method wherein the somatic cells themselves to be
reprogrammed are used instead of the feeder cells (Takahashi K, et
al. (2009), PLoS One. 4:e8067 or WO2010/137746), and a method
wherein an extracellular matrix (e.g., Laminin-5 (WO2009/123349) or
Matrigel (BD)) is used instead.
[0053] Other examples include a method wherein the culture is
carried out using a serum-free medium (Sun N, et al. (2009), Proc
Natl Acad Sci U S A. 106:15720-15725). Further, in order to enhance
the establishment efficiency, iPS cells may be established under
low oxygen conditions (at an oxygen concentration of 0.1% to 15%)
(Yoshida Y, et al. (2009), Cell Stem Cell. 5:237-241 or
WO2010/013845).
[0054] During the culture, the medium is replaced with a fresh
medium once every day from Day 2 of the culture. The number of
somatic cells used for nuclear reprogramming is not restricted, and
usually within the range of about 5.times.10.sup.3 to about
5.times.10.sup.6 cells per 100-cm.sup.2 area on the culture
dish.
[0055] iPS cells may be selected based on the shape of each formed
colony. In cases where a drug resistance gene is introduced as a
marker gene such that the drug resistance gene is expressed in
conjunction with a gene that is expressed when a somatic cell was
reprogrammed (e.g., Oct3/4 or Nanog), the established iPS cells can
be selected by culturing the cells in a medium containing the
corresponding drug (selection medium). Further, iPS cells can be
selected by observation under a fluorescence microscope in cases
where the marker gene is the gene of a fluorescent protein; by
adding a luminescent substrate in cases where the marker gene is
the gene of luciferase; or by adding a coloring substrate in cases
where the marker gene is the gene of a coloring enzyme.
[0056] The term "somatic cells" used in the present description
means any animal cells (preferably cells of a mammal including
human) excluding germ-line cells and totipotent cells such as eggs,
oocytes and ES cells. Examples of the somatic cells include, but
are not limited to, any of fetal somatic cells, neonatal somatic
cells, and mature, healthy and diseased somatic cells, as well as
any of primary cultured cells, subcultured cells and established
cell lines. Specific examples of the somatic cells include (1)
tissue stem cells (somatic stem cells) such as neural stem cells,
hematopoietic stem cells, mesenchymal stem cells and dental pulp
stem cells; (2) tissue progenitor cells; and (3) differentiated
cells such as lymphocytes, epithelial cells, endothelial cells,
muscle cells, fibroblasts (skin cells and the like), hair cells,
hepatic cells, gastric mucosal cells, enterocytes, spleen cells,
pancreatic cells (pancreatic exocrine cells and the like), brain
cells, lung cells, kidney cells and adipocytes.
[0057] In cases where iPS cells are used as a material for the
cells to be transplanted, somatic cells having the same or
substantially the same HLA genotype as that of the individual to
which the cells are to be transplanted are preferably used in view
of prevention of the rejection reaction. Here, "substantially the
same" means that the HLA genotype is matching to an extent at which
the immune reaction against the transplanted cells can be
suppressed with an immunosuppressive agent. For example, the
somatic cells have matched HLA types at 3 loci, HLA-A, HLA-B and
HLA-DR, or at 4 loci further including HLA-C.
(E) ES Cells Derived from Cloned Embryo Obtained by Nuclear
Transfer
[0058] ntES cells are ES cells derived from a cloned embryo
prepared by the nuclear transfer technique, and have properties
which are almost the same as those of ES cells derived from
fertilized eggs (T. Wakayama et al. (2001), Science, 292:740-743;
S. Wakayama et al. (2005), Biol. Reprod., 72:932-936; J. Byrne et
al. (2007), Nature, 450:497-502). That is, an ntES (nuclear
transfer ES) cell is an ES cell established from the inner cell
mass of a blastocyst derived from a cloned embryo obtained by
replacement of the nucleus of an unfertilized egg with the nucleus
of a somatic cell. For preparation of an ntES cell, the combination
of the nuclear transfer technique (J. B. Cibelli et al. (1998),
Nature Biotechnol., 16:642-646) and the ES cell preparation
technique (described above) is employed (Sayaka Wakayama et al.
(2008), Experimental Medicine 26(5) (extra edition):47-52). In
nuclear transfer, reprogramming can be achieved by injecting the
nucleus of a somatic cell into a mammalian enucleated unfertilized
egg and culturing the resultant for several hours.
<Method for Preparing Cardiomyocytes from Pluripotent Stem
Cells>
[0059] The method for inducing differentiation of pluripotent stem
cells into cardiomyocytes is not restricted, and, for example, the
following method may be used.
[0060] Pluripotent stem cells may be separated by an arbitrary
method and subjected to suspension culture, or adherent culture
using a coated culture dish. In the method of separation, the cells
may be mechanically separated, or may be separated using an EDTA
solution (e.g., 0.5 mM EDTA solution or Versene (Invitrogen)),
separation solution having protease activity and collagenase
activity (e.g., Accutase (.TM.) or Accumax (.TM.)), or separation
solution having only collagenase activity. In the suspension
culture, the culture dish may have either a surface which is not
subjected to artificial treatment for the purpose of enhancing
adhesiveness to cells such as coating treatment with an
extracellular matrix or the like, or a surface which is
artificially treated such that adhesion is suppressed (for example,
by coating treatment with polyhydroxyethylmethacrylate (poly-HEMA).
In the adherent culture, the culture dish may be one coated with
Matrigel (BD), type I collagen, type IV collagen, gelatin, laminin,
heparan sulfate proteoglycan or entactin, or a combinations
thereof
[0061] The suspension culture and the adherent culture may be
performed in combination. In an embodiment of the present invention
where these are performed in combination, the suspension culture
may be followed by adherent culture without any treatment, or
mesodermal cells prepared by the suspension culture may be selected
before the adherent culture. In the present description, "mesoderm"
includes germ layers constituted by cells capable of producing,
during development, the body cavity and mesothelium lining it,
muscles, skeletons, dermis, connective tissues, heart/blood vessels
(including vascular endothelium), blood (including blood cells),
lymph vessels and spleen, kidney and ureter, and gonads (testis,
uterus and gonadal epithelium). These can be detected by expression
of markers such as T, KDR, FOXF1, FLK1 and/or BMP4. The cells
preferably express KDR or FLK1.
[0062] The adherent culture may be carried out by co-culture with
feeder cells. Examples of the feeder cells to be used in the
co-culture include OP9 cells (Nishikawa, S. I. et al., Development
125, 1747-1757 (1998)) and END-2 cells (Mummery C, et al.,
Circulation. 107:2733-40 (2003)), but, in cases where the cultured
cells are used as a material for cells to be transplanted,
co-culture is preferably avoided in view of prevention of
contamination with other types of cells.
[0063] In another mode, embryoid bodies (EBs) are formed from
pluripotent stem cells isolated by an arbitrary method, and then
subjected to adherent culture in an arbitrary medium in a coated
culture dish. In other mode, EBs are formed from pluripotent stem
cells isolated by an arbitrary method, and then subjected to
adherent culture in an arbitrary medium in a coated culture dish,
followed by again the formation of EBs. By these, cardiomyocytes
can be induced. The EBs are usually formed by suspension culture,
but the mode is not limited thereto.
[0064] In this process, the medium may be prepared using, as a
basal medium, a medium for use in animal cell culture. Examples of
the basal medium include IMDM medium, Medium 199, Eagle's Minimum
Essential Medium (EMEM), a-MEM medium, Dulbecco's modified Eagle's
Medium (DMEM), Ham's F12 medium, RPMI 1640 medium and Fischer's
medium, and mixtures thereof The medium is preferably StemPro 34
medium. The medium may either contain serum or be serum-free. The
culture medium may also contain, as required, one or more of serum
replacements such as albumin, transferrin, Knockout Serum
Replacement (KSR) (serum replacement for FBS in ES cell culture),
fatty acids, insulin, collagen precursors, trace elements,
2-mercaptoethanol, 3'-thiolglycerol and ITS-supplements; and/or one
or more of substances such as B27 supplement, N2 supplement,
lipids, amino acids, L-glutamic acid, Glutamax (Invitrogen),
non-essential amino acids, vitamins, growth factors, cytokines, Wnt
inhibitors, antibiotics, antioxidants, pyruvic acid, buffers and
inorganic salts. Examples of the cytokines include activin A, BMP4,
b-FGF, and VEGF. Examples of Wnt inhibitors include DKK1 (NCBI
Accession No. NM.sub.--012242 in human), and Sclerostin (NCBI
Accession No. NM.sub.--025237 in human)IWR-1 (Merck Millipore),
IWP-2 (Sigma-Aldrich), IWP-3 (Sigma-Aldrich), IWP-4
(Sigma-Aldrich), PNU-74654 (Sigma-Aldrich), XAV939 (Sigma-Aldrich),
and derivatives thereof.
[0065] In cases where the substances described above are added to
the medium, different agents may be added at each stage of culture.
The concentrations of the substances may be arbitrarily set
depending on the type of the cells to which the agents are
applied.
[0066] The culture temperature is not restricted and may be about
30 to 40.degree. C., preferably about 37.degree. C., and the
culture is carried out under an atmosphere of CO.sub.2-containing
air wherein the CO.sub.2 concentration is preferably about 2 to 5%.
The culture is carried out for a number of days required for
cardiac troponin and/or aMHC to be expressed, and the culturing
period is, for example, not less than 8 days.
[0067] Induction of differentiation into cardiomyocytes may be
carried out under low oxygen conditions. The oxygen concentration
in the low oxygen conditions is, for example, 1 to 10%, preferably
5%, but the concentration is not limited thereto. The period under
low oxygen conditions is not limited as long as induction of
differentiation into cardiomyocytes can be achieved therewith, and
may be, for example, 1 to 20 days, preferably 12 days.
[0068] For example, the method for producing cardiomyocytes from
pluripotent stem cells is carried out under low oxygen conditions
where pluripotent stem cells are cultured in StemPro 34
supplemented with BMP4 and ROCK inhibitor for 24 hours to allow
formation of EBs, followed by 3 days of culture in a medium
supplemented with activin A, BMP4 and bFGF, 4 days of culture in a
medium supplemented with VEGF and DKK-1, and then several days of
culture in a medium supplemented with VEGF and b-FGF. The last
culture step may be carried out for an arbitrary period in order to
evaluate the cells using a cardiomyocyte marker(s).
[0069] The thus produced cardiomyocytes may be either a population
constituted by a single type cells, or may be a cell population
that also comprises another type of cells.
<Method for Extraction or Detection of Cardiomyocytes>
[0070] For extraction or detection of cardiomyocytes from a cell
population containing cardiomyocytes, any reagent having specific
affinity to NCAM1, SSEA3, SSEA4 or CD340 may be used, and, for
example, an antibody, aptamer, peptide or compound that
specifically recognizes such a protein may be used. The reagent is
preferably an antibody or a fragment thereof.
[0071] The antibody may be either a polyclonal antibody or a
monoclonal antibody. These antibodies may be prepared using a
technique well known to those skilled in the art (Current protocols
in Molecular Biology edit. Ausubel et al. (1987) Publish. John
Wiley and Sons. Section 11.12-11.13). More specifically, in cases
where the antibody is a polyclonal antibody, a protein encoded by
NCAM1, SSEA3, SSEA4 or CD340 which was expressed in E. coli or the
like and purified according to a conventional method, or a
synthetic oligopeptide having a partial amino acid sequence of the
protein, may be used to immunize a nonhuman animal such as a
rabbit, followed by obtaining the antibody from serum of the
immunized animal according to a conventional method. On the other
hand, the monoclonal antibody can be obtained from hybridoma cells
prepared by cell fusion of spleen cells obtained from the
above-described immunized nonhuman animal with myeloma cells
(Current protocols in Molecular Biology edit. Ausubel et al. (1987)
Publish. John Wiley and Sons. Section 11.4-11.11). Examples of the
fragment of the antibody include parts (for example, the Fab
fragment) of the antibody and synthetic antibody fragments (for
example, the single-chain Fv fragment "ScFv"). Antibody fragments
such as Fab and F(ab).sub.2 may also be prepared according to
methods well known in the field of genetic engineering.
[0072] In order to distinguish and separate cells to which the
reagent having affinity is bound, the reagent may be bound or
conjugated to a detectable substance such as a fluorescent label,
radioactive label, chemiluminescent label, enzyme, biotin or
streptavidin, or to a substance that enables isolation and
extraction of the cells, such as protein A, protein G, beads or
magnetic beads.
[0073] The reagent having affinity may also be indirectly labeled.
The labeling can be carried out by various methods known to those
skilled in the art, and examples of the methods include a method
using a preliminarily labeled antibody (secondary antibody) that
specifically binds to an antibody against NCAM1, SSEA3, SSEA4 or
CD340.
[0074] Examples of the method for detecting cardiomyocytes include
flow cytometry and a method wherein the cells are isolated and
purified, followed by detection of the cells (for example, using a
protein chip).
[0075] Examples of the method for extracting cardiomyocytes include
a method wherein a large particle is conjugated to the reagent
having affinity to cause precipitation, a method wherein cells are
sorted using magnetic beads by the magnetism (e.g., MACS), a method
wherein a fluorescent label is used to employ a cell sorter, and a
method wherein a carrier to which an antibody or the like is
immobilized (e.g., cell-concentrating column) is used.
[0076] The present invention will now be described more concretely
by way of Examples below, but the present invention is not
restricted to these Examples.
EXAMPLES
Example 1
Preparation of Pluripotent Stem Cells
[0077] The 201B7-M6GIP4 strain and the 606A1 strain, which show
cardiomyocyte-specific expression of EGFP, were prepared as
described below. The 201 B7-M6GIP4 strain and the 606A1 strain were
cultured by a conventional method
[0078] (Takahashi K, et al. Cell. 131: 861-72, 2007 and Nakagawa M,
et al. Nat Biotechnol. 26: 101-6, 2008).
(1) 201B7-M6GIP4 Strain
[0079] OCT3/4, SOX2,KLF4 and C-MYC were introduced to human
fibroblasts using a retrovirus, and the resulting cells were
cultured on mitomycin-treated SNL feeder cells to establish iPS
cells (201B7 strain). The promoter region of the human MYH6 gene
and a sequence of the EGFP gene were introduced to the resulting
201B7 strain, to prepare a cell line (201 B7-M6GIP4 strain). The
201 B7-M6GIP4 strain has been confirmed to show
cardiomyocyte-specific expression of GFP after induction of
differentiation.
(2) 606A1 Strain
[0080] An episomal vector set (pCXLE-hOct3/4-shp53-F, pCXLE-hSK and
pCXLE-hUL) that expresses OCT3/4, SOX2, KLF4, LIN28, L-MYC and p53
shRNAi was introduced to human umbilical cord blood cells by
electroporation, and the resulting cells were cultured on
mitomycin-treated mouse embryonic fibroblast feeders, to prepare an
iPS cell line (606A1 strain).
[0081] The above-described cells of (1) and (2) were cultured on a
6-cm dish to 80 to 90% confluence. The feeder cells were removed,
and the cultured cells were plated on a Matrigel-coated dish using
trypsin/EDTA and cultured for additional 3 to 4 days. The cells
were then detached by addition of collagenase B and a trypsin/EDTA
solution.
Example 2
Method for Induction of Cardiomyocytes
[0082] The human iPS cells were plated on a dish at a density of
1.5 to 2.0.times.10.sup.6 cells per well, and cultured under low
oxygen conditions (5%) in StemPro 34 medium supplemented with 10
ng/ml BMP4 and 10 .mu.M ROCK inhibitor for 24 hours to allow
formation of EBs. On the following day, the medium was replaced
with a medium supplemented with 6 ng/ml activin A (R & D
Systems), 10 ng/ml BMP4 (R & D Systems) and 5 ng/ml bFGF (R
& D Systems), and the culture was further continued for 3 days.
Subsequently, the medium was replaced with a medium supplemented
with 10 ng/ml VEGF (R & D Systems) and 150 ng/ml DKK-1 (R &
D Systems), and the culture was further continued for 4 days. The
medium was then replaced with a medium supplemented with 10 ng/ml
VEGF (R & D Systems) and 5 ng/ml bFGF (R & D Systems), and
the culture was further continued for 52 days. The cells at each
step of differentiation from the iPS cells into cardiomyocytes were
evaluated with the cardiomyocyte markers described below.
Example 3
Evaluation of Cells with Cardiomyocyte Marker NCAM1
[0083] The human iPS cell (201B7-M6GIP4 strain and 606A1
strain)-derived cardiomyocytes induced by the method described in
Example 2 were stained with an anti-NCAM1 antibody (BD) on Day 0,
Day 4, Day 8, Day 12, Day 16, Day 20, Day 26, Day 33, Day 45 and
Day 60 after the induction, and evaluated using flow cytometry
(FIG. 2). As a result of cardiomyocyte purification based on the
difference in expression of NCAM1 on Day 30, the ratio of troponin
T-positive cardiomyocytes, which was 58.5% before the purification,
became 88.6% and 71.3% in NCAM1-highly expressing cells and
NCAM1-moderately expressing cells, respectively, obtained by
sorting by flow cytometry (FIG. 3). Thus, highly pure
cardiomyocytes were obtained.
[0084] The above results showed that cardiomyocytes can be
identified by using NCAM1 as an index of differentiation into
cardiomyocytes.
Example 4
Evaluation of Cells with Cardiomyocyte Marker SSEA3
[0085] The human iPS cell (201B7-M6GIP4 strain and 606A1
strain)-derived cardiomyocytes induced by the method described in
Example 2 were stained with an anti-SSEA3 antibody (BD) on Day 0,
Day 4, Day 8, Day 12, Day 16, Day 20, Day 26, Day 33, Day 45 and
Day 60 after the induction, and evaluated using flow cytometry. As
a result, immature cardiomyocytes on Day 8 were negative for the
marker, but high expression was observed in a part of MYH6-positive
cardiomyocytes on Day 16 to Day 26 (FIG. 4).
[0086] As a result of cardiac muscle purification based on the
difference in expression of SSEA3 on Day 16, the ratio of
MYH6-positive cells, which was 69.7% before the purification,
became as high as 81.5% after the purification.
[0087] The above results showed that cardiomyocytes can be
identified by using SSEA3 as an index of differentiation into
cardiomyocytes.
Example 5
Evaluation of Cells with Cardiomyocyte Marker SSEA4
[0088] The human iPS cell (201B7-M6GIP4 strain and 606A1
strain)-derived cardiomyocytes induced by the method described in
Example 2 were stained with an anti-SSEA4 antibody (BD) on Day 0,
Day 4, Day 8, Day 12, Day 16, Day 20, Day 26, Day 33, Day 45 and
Day 60 after the induction, and evaluated using flow cytometry. As
a result, immature cardiomyocytes on Day 8 were negative for the
marker, but high expression was observed in many of MYH6-positive
cardiomyocytes on Day 12 and later, and high expression was
observed in most of MYH6-positive cardiomyocytes especially on Day
12 to Day 33(FIG. 5).
[0089] As a result of cardiac muscle purification based on the
difference in expression of SSEA4 on Day 16, the ratio of
MYH6-positive cells, which was 69.7% before the purification,
became as high as 84.0% after the purification.
[0090] The above results showed that cardiomyocytes can be
identified by using SSEA4 as an index of differentiation into
cardiomyocytes.
Example 6
Evaluation of Cells with Cardiomyocyte Marker CD340
[0091] The human iPS cell (201B7-M6GIP4 strain and 606A1
strain)-derived cardiomyocytes induced by the method described in
Example 2 were stained with an anti-CD340 antibody (BD) on Day 0,
Day 4, Day 8, Day 12, Day 16, Day 20, Day 30 and Day 45 after the
induction, and evaluated using flow cytometry. As a result,
MYH6-positive cardiomyocytes were CD340-positive on Day 8 and later
after the induction (FIG. 6). As a result of cardiac muscle
purification based on the difference in expression of CD340 on Day
16 and Day 30, the ratio of MYH6-positive cells on Day 16, which
was 35.7% before the purification, became 71.2% after the
purification, and the ratio on Day 30, which was 36.7% before the
purification, became 65.7% after the purification.
[0092] The above results showed that cardiomyocytes can be
identified by using CD340 as an index of differentiation into
cardiomyocytes.
INDUSTRIAL APPLICABILITY
[0093] According to the present invention, cardiomyocytes can be
efficiently extracted or detected from a cell population comprising
the cardiomyocytes.
[0094] While the present invention has been described with emphasis
on preferred embodiments, it is obvious to those skilled in the art
that the preferred embodiments can be modified. The present
invention intends that the present invention can be embodied by
methods other than those described in detail in the present
specification. Accordingly, the present invention encompasses all
modifications encompassed in the gist and scope of the appended
"CLAIMS."
[0095] The contents disclosed in any publication cited herein,
including patents and patent applications, are hereby incorporated
in their entireties by reference, to the extent that they have been
disclosed herein.
* * * * *