U.S. patent application number 13/637107 was filed with the patent office on 2013-08-01 for composition for treatment of damaged part.
This patent application is currently assigned to NATIONAL UNIVERSITY CORPORATION NAGOYA UNIVERSITY. The applicant listed for this patent is Katsumi Ebisawa, Hisashi Hattori, Takanori Inoue, Kohki Matsubara, Kiyoshi Sakai, Masahiko Sugiyama, Minoru Ueda, Yoichi Yamada, Akihito Yamamoto. Invention is credited to Katsumi Ebisawa, Hisashi Hattori, Takanori Inoue, Kohki Matsubara, Kiyoshi Sakai, Masahiko Sugiyama, Minoru Ueda, Yoichi Yamada, Akihito Yamamoto.
Application Number | 20130195991 13/637107 |
Document ID | / |
Family ID | 44673335 |
Filed Date | 2013-08-01 |
United States Patent
Application |
20130195991 |
Kind Code |
A1 |
Ueda; Minoru ; et
al. |
August 1, 2013 |
Composition for Treatment of Damaged Part
Abstract
The present invention provides a damaged part treatment
composition for repairing a damaged part of a target tissue that
includes a stem cell-conditioned medium obtained by culturing stem
cells; a damaged part treatment method for repairing or restoring a
damaged part of a target tissue that includes administering the
damaged part treatment composition to a patient having the target
tissue for the damaged part treatment composition in an amount
therapeutically effective for repairing the damaged part of the
target tissue; a method of treating cerebral infarction that
includes administering the damaged part treatment composition to a
cerebral infarct patient in an amount effective for repairing a
damaged part of the brain; and a method of treating a CNS disease
that includes administering, as a CNS disease treatment
composition, the damaged part treatment composition to a CNS
disease patient in a therapeutically effective amount.
Inventors: |
Ueda; Minoru; (Nagoya-shi,
JP) ; Yamada; Yoichi; (Nagoya-shi, JP) ;
Ebisawa; Katsumi; (Nagoya-shi, JP) ; Yamamoto;
Akihito; (Nagoya-shi, JP) ; Sakai; Kiyoshi;
(Nagoya-shi, JP) ; Matsubara; Kohki; (Nagoya-shi,
JP) ; Hattori; Hisashi; (Nagoya-shi, JP) ;
Sugiyama; Masahiko; (Nagoya-shi, JP) ; Inoue;
Takanori; (Nagoya-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ueda; Minoru
Yamada; Yoichi
Ebisawa; Katsumi
Yamamoto; Akihito
Sakai; Kiyoshi
Matsubara; Kohki
Hattori; Hisashi
Sugiyama; Masahiko
Inoue; Takanori |
Nagoya-shi
Nagoya-shi
Nagoya-shi
Nagoya-shi
Nagoya-shi
Nagoya-shi
Nagoya-shi
Nagoya-shi
Nagoya-shi |
|
JP
JP
JP
JP
JP
JP
JP
JP
JP |
|
|
Assignee: |
NATIONAL UNIVERSITY CORPORATION
NAGOYA UNIVERSITY
Nagoya-shi
JP
|
Family ID: |
44673335 |
Appl. No.: |
13/637107 |
Filed: |
March 25, 2011 |
PCT Filed: |
March 25, 2011 |
PCT NO: |
PCT/JP2011/057412 |
371 Date: |
December 13, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61317713 |
Mar 26, 2010 |
|
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61410370 |
Nov 5, 2010 |
|
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61437697 |
Jan 31, 2011 |
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Current U.S.
Class: |
424/572 ;
514/8.1; 514/8.2; 514/8.5; 514/8.9; 514/9.5 |
Current CPC
Class: |
A61K 38/18 20130101;
A61P 19/08 20180101; A61P 25/00 20180101; A61P 17/00 20180101; A61K
35/12 20130101; A61K 35/545 20130101; A61K 38/30 20130101; A61P
27/02 20180101; A61K 38/1858 20130101; A61P 1/02 20180101; A61P
17/02 20180101; C12N 5/0664 20130101; A61P 25/16 20180101; A61K
38/1866 20130101; A61K 35/32 20130101; A61K 35/30 20130101; A61P
25/28 20180101; A61K 35/28 20130101; A61P 9/10 20180101; A61K
38/1833 20130101; A61P 25/14 20180101; A61K 38/1841 20130101; A61K
38/18 20130101; A61K 2300/00 20130101; A61K 35/28 20130101; A61K
2300/00 20130101; A61K 35/32 20130101; A61K 2300/00 20130101; A61K
35/545 20130101; A61K 2300/00 20130101; A61K 35/30 20130101; A61K
2300/00 20130101; A61K 38/30 20130101; A61K 2300/00 20130101; A61K
38/1833 20130101; A61K 2300/00 20130101; A61K 38/1841 20130101;
A61K 2300/00 20130101; A61K 38/1858 20130101; A61K 2300/00
20130101; A61K 38/1866 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/572 ;
514/8.5; 514/8.1; 514/8.2; 514/9.5; 514/8.9 |
International
Class: |
A61K 35/12 20060101
A61K035/12 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 1, 2010 |
JP |
2010-267962 |
Feb 23, 2011 |
JP |
2011-037028 |
Claims
1. A damaged part treatment composition for repairing a damaged
part of a target tissue, the composition comprising a stem
cell-conditioned medium obtained by culturing stem cells.
2. The damaged part treatment composition according to claim 1,
which does not comprise the stem cells.
3. The damaged part treatment composition according to claim 1,
wherein the stem cell-conditioned medium comprises at least two
cytokines.
4. The damaged part treatment composition according to claim 1,
wherein the stem cell-conditioned medium comprises at least two
cytokines selected from the group consisting of vascular
endothelial growth factor (VEGF), hepatocyte growth factor (HGF),
insulin-like growth factor (IGF), platelet-derived growth factor
(PDGF) and transforming growth factor .beta. (TGF-.beta.).
5. The damaged part treatment composition according to claim 1,
wherein the stem cells are somatic stem cells.
6. The damaged part treatment composition according to claim 1,
wherein the stem cells are derived from mesenchymal stem cells.
7. The damaged part treatment composition according to claim 1,
wherein the stem cells are dental pulp stem cells.
8. The damaged part treatment composition according to claim 1,
which does not comprise any serum.
9. The damaged part treatment composition according to claim 1,
wherein the treatment of a damaged part comprises treatment of
damage to skin, periodontal tissue or bone, treatment of cerebral
infarction, or treatment of a central nervous system (CNS)
disease.
10. The damaged part treatment composition according to claim 1,
wherein the treatment of a damaged part comprises treatment of a
CNS disease, and the CNS disease is a disease or disorder selected
from the group consisting of a spinal cord injury, a
neurodegenerative disorder, degeneration or loss of neuronal cells
and a retinal disease involving a neuronal cell disorder.
11. A method of producing the damaged part treatment composition of
claim 1, the method comprising the following steps (1) to (3): (1)
a step of selecting adhesive cells from dental pulp cells; (2) a
step of culturing the adhesive cells; and (3) a step of collecting
a conditioned medium.
12. The production method according to claim 11, further comprising
the following step (4): (4) a step of subjecting the collected
conditioned medium to at least one treatment selected from the
group consisting of centrifugation, concentration, solvent
substitution, dialysis, freezing, drying, freeze-drying, dilution,
desalting and storage.
13. The production method according to claim 11, further comprising
one of the following steps (a) or (b): (a) a step of checking the
collected conditioned medium with respect to the presence or
absence of a neurite outgrowth activity in the presence of a nerve
regeneration inhibitory substance; or (b) a step of checking the
collected conditioned medium with respect to the presence or
absence of an apoptosis inhibitory activity toward neuronal
cells.
14. A damaged part treatment method for repairing a damaged part of
a target tissue, the method comprising administering the damaged
part treatment composition of claim 1 to a patient having the
target tissue for the damaged part treatment composition, in an
amount effective for repairing the damaged part of the target
tissue.
15. The damaged part treatment method according to claim 14,
wherein the repair of the damaged part is achieved based on an
ability of endogenous stem cells.
16. The damaged part treatment method according to claim 14,
wherein the damaged part treatment composition is administered by
an administration method selected from the group consisting of
intravenous administration, intraarterial administration,
intraportal administration, intradermal administration,
subcutaneous administration, intramuscular administration,
intraperitoneal administration and intranasal administration.
17. A method of treating cerebral infarction comprising
administering the damaged part treatment composition of claim 1 to
a cerebral infarction patient, in an amount therapeutically
effective for repairing a damaged part of the brain.
18. The method of treating cerebral infarction according to claim
17, wherein the damaged part treatment composition is administered
by intranasal administration.
19. A method of treating a CNS disease comprising administering the
damaged part treatment composition of claim 1 as a CNS disease
treatment composition to a CNS disease patient, in a
therapeutically effective amount.
20. The treatment method according to claim 19, wherein a dental
pulp stem cell is administered to the CNS disease patient
simultaneously with, or subsequently to, the administration of the
CNS disease treatment composition.
21. The treatment method according to claim 20, wherein the dental
pulp stem cell is an undifferentiated dental pulp stem cell that
has not been subjected to differentiation-inducing treatment after
acquisition thereof, or a differentiation-induced dental pulp stem
cell that has been induced to differentiate into a neural cell
after acquisition thereof.
22. The treatment method according to claim 19, wherein a
pluripotent stem cell that has been induced to differentiate into a
neural cell is administered to the CNS disease patient after the
administration of the CNS disease treatment composition.
23. A method of determining whether or not a prepared dental pulp
stem cell-conditioned medium is effective as an active ingredient
of the CNS disease treatment composition to be employed in the CNS
disease treatment method according to claim 19, the method
comprising at least one of the following steps (a) or (b): (a) a
step of checking the conditioned medium with respect to the
presence or absence of a neurite outgrowth activity in the presence
of a nerve regeneration inhibitory substance; or (b) a step of
checking the conditioned medium with respect to the presence or
absence of an apoptosis inhibitory activity toward neuronal cells.
Description
TECHNICAL FIELD
[0001] The present invention relates to a composition for treatment
of a damaged part, and a treatment method using the same.
[0002] Regenerative medicine utilizing stem cells attracts
attention as a versatile alternative technique for diseases that
are hard to treat by conventional medicine.
[0003] Regenerative medicine using stem cells is a promising tool
in a new clinical platform for a whole spectrum of intractable
diseases. Various stem cells have been reported, including
embryonic stem cells (ES cells), induced pluripotent stem cells
(iPS cells) and somatic stem cells. Among somatic stem cells,
mesenchymal stem cells (MSCs) isolated from various tissues
including bone marrow, adipose tissue, skin, umbilical cord and
placenta have been used in particular in clinical applications in
skin regeneration. However, bone marrow aspiration is an invasive
and painful procedure for the donor. In addition, the number, and
proliferation and differentiation potential of bone marrow stem
cells (BMSCs) decline with increasing age.
[0004] There are many diseases to which regenerative medicine is
applicable or expected to be applicable, and various researches for
clinical application thereof have been carried out. A neurological
disorder, particularly an intractable neurological disorder such as
spinal cord injury, is one of the diseases to which therapy by
regenerative medicine is expected to be applied.
[0005] Transplantation therapy of an intractable neurological
disorder using neural stem cells from human embryos or ES cells
(for example, Japanese Patent Application Laid-open (JP-A) No.
2002-281962) is recognized as a realistic research target, but has
a serious problem in terms of morality and safety. Therefore,
practical "stem cell source" is still searched for (for example,
Keirstead et al., Human embryonic stem cell-derived oligodendrocyte
progenitor cell transplants remyelinate and restore locomotion
after spinal cord injury, Journal of Neuroscience (2005) vol. (19)
pp. 4694; Okano et al., Neural stem cells and regeneration of
injured spinal cord, Kidney international (2005), Vol. 68, pp.
1927-1931; Okada et al., Spatiotemporal recapitulation of central
nervous system development by murine embryonic stem cell derived
neural stem and progenitor cells, Stem Cells (2008) vol. 26 (12)
pp. 3086-3098).
[0006] Examples of stem cells in a living organism include stem
cells derived from bone marrow or adipose tissue (for example,
International Publication 02/086108 pamphlet). These stem cells
have shortcomings such as (1) reduction with age in the number of
stem cells that can be obtained, (2) difficulty in terms of
ensuring the safety of transplanted stem cells due to accumulation
of genetic mutations with age, (3) low proliferative capacity of
the cells and (4) severe body invasion accompanying the collection
of stem cells (for example, Gronthos et al., Postnatal human dental
pulp stem cells (DPSCs) in vitro and in vivo, Proc Natl Acad Sci
USA (2000) vol. 97 (25) pp. 13625-13630; Miura et al., SHED: stem
cells from human exfoliated deciduous teeth, Proceedings of the
National Academy of Sciences (2003) Vol. 100, 5807-5812).
Development of a novel stem cell resource for treatment of
intractable neurological disorders to solve these problems is
important.
[0007] Stem cells from human exfoliated deciduous teeth (SHED) and
permanent teeth dental pulp stem cells (DPSCs) derived from wisdom
tooth, which are medical wastes, were identified as novel stem cell
groups having self-renewal capacity and pluripotency similar to
those of BMSCs.
[0008] These cells are cell groups derived from neural crest, and
exhibit similar properties to neural lineage and high reactivity to
the induction of neuronal differentiation (for example, Miura et
al., SHED: stem cells from human exfoliated deciduous teeth,
Proceedings of the National Academy of Sciences (2003) Vol. 100,
5807-5812; Arthur et al., Adult human dental pulp stem cells
differentiate toward functionally active neurons under appropriate
environmental cues, Stem Cells (2008) vol. 26 (7) pp. 1787-1795).
Since SHED and DPSCs are self-derived tissue stem cells, safety in
the case of transplantation is high, and hardly any moral problem
is involved.
[0009] However, in conventional study on SHED and DPSCs, there is
no finding more than fragmentary analysis of neuronal cell lineage,
or observation of engraftment of neuronal-differentiation-induced
SHED or DPSCs transplanted into rodents (for example, Arthur et
al., Adult human dental pulp stem cells differentiate toward
functionally active neurons under appropriate environmental cues,
Stem Cells (2008) vol. 26 (7) pp. 1787-1795; Huang et al., Putative
dental pulp-derived stem/stromal cells promote proliferation and
differentiation of endogenous neural cells in the hippocampus of
mice, Stem Cells (2008) vol. 26 (10) pp. 2654-2663).
[0010] Further, there are reports that DPSCs have a potential to be
employein cell-based treatment for systemic disorders such as
neuronal disorders and cardiac diseases, and that DPSCs ameliorate
ischemic disorders (Arthur et al., Adult human dental pulp stem
cells differentiate toward functionally active neurons under
appropriate environmental cues, Stem Cells (2008) vol. 26 (7) pp.
1787-1795; Gandia C, Arminan A, Garcia-Verdugo J M, et al., Human
dental pulp stem cells improve left ventricular function, induce
angiogenesis, and reduce infarct size in rats with acute myocardial
infarct, Stem Cells 2008; 26: 638-645; Iohara K, Zheng L, Wake H,
et al., A novel stem cell source for vasculogenesis in schemia:
subfraction of side population cells from dental pulp, Stem Cells
2008; 26:2408-2418).
[0011] Recent research has indicated that MSCs can contribute to
skin repair. Further, wound healing by external application of
various growth factors is widely studied. However, the result of
the use of growth factors in single administration or multiple
administrations, or the result of the use of multiple growth
factors in combination with a view to obtaining synergistic
effects, has not been clinically confirmed.
[0012] Further, treatment of a group of aged population who has
excessively been exposed to sunlight is a large focus of
cosmeceutical products and dermatologists. Various non-invasive
treatments and topical cosmeceutical products are used in order to
treat some of the symptoms of photo-aged skin such as wrinkles
(Chung J H, Youn S H, Kwon O S, Cho K H, Youn J I, Eun H C.,
Regulations of collagen synthesis by ascorbic acid, transforming
growth factor-beta and interferongamma in human dermal fibroblasts
cultured in three-dimensional collagen gel are photoaging- and
aging-independent, J Dermatol Sci 1997; 15: 188-200; Fitzpatrick R
E, Rostan E F., Reversal of photodamage with topical growth
factors: a pilot study, J Cosmet Laser Ther 2003; 5: 25-34).
[0013] Exposure to short-wavelength ultraviolet rays (UVB), which
is one reason of aging, is known to stimulate collagenase
production by human dermal fibroblasts (HDF) in the dermis, and to
up-regulate the expression of a collagenase gene. This is
considered to induce the degradation of collagen and deposition of
a degenerated elastic tissue which appears as skin wrinkles and
yellowing.
[0014] Previous studies indicate that MSCs produce various
cytokines such as vascular endothelial growth factor (VEGF),
hepatocyte growth factor (HGF), insulin-like growth factor (IGF),
platelet-derived growth factor (PDGF) and transforming growth
factor .beta. (TGF-.beta.). In recent years, the production and
secretion of cytokines are reported as important functions of MSCs,
and a wide variety of pharmaceutical activities of MSCs has been
demonstrated in, particularly, skin biology (Jettanacheawchankit S,
Sasithanasate S, Sangvanich P, Banlunara W, Thunyakitpisal P.,
Acemannan stimulates gingival fibroblast proliferation; expressions
of keratinocyte growth factor-1, vascular endothelial growth
factor, and type I collagen; and wound healing, J Pharmacol Sci.
2009 April; 109(4): 525-531; Miura et al., SHED: stem cells from
human exfoliated deciduous teeth, Proceedings of the National
Academy of Sciences (2003) Vol. 100, 5807-5812; Safavi S M, Kazemi
B, Esmaeili M, Fallah A, Modarresi A, Mir M., Effects of low-level
He--Ne laser irradiation on the gene expression of IL-1beta,
TNF-alpha, IFN-gamma, TGF-beta, bFGF, and PDGF in rat's gingiva,
Lasers Med. Sci. 2008 July; 23(3): 331-335; Saygun I, Karacay S,
Serdar M, Ural A U, Sencimen M, Kurtis B, Effects of laser
irradiation on the release of basic fibroblast growth factor
(bFGF), insulin like growth factor-1 (IGF-1), and receptor of IGF-1
(IGFBP3) from gingival fibroblasts, Lasers Med. Sci. 2008 April;
23(2): 211-215). For example, it was reported that MSCs have skin
healing effects via production of various growth factors (see,
Minoru Ueda, The Use of fibroblasts, The Biochemical Society,
11-15, 2007). These growth factors activated HDF, enhanced
growth/migration of HDF, and mediated collagen secretion from HDF.
Since secretion factors from MSCs were indicated to protect HDF
from oxidation stress, the antioxidant effect of MSCs was also
demonstrated. Application of topical growth factors resulted in
stimulation of repair of photoaging of the face, and provided a
smoother clinical appearance of the skin with de novo synthesis of
collagen, decreased thickening of epithelium and a reduction in
visually-noticeable wrinkles (He H, Yu J, Liu Y, et al., Effects of
FGF2 and TGFbeta1 on the differentiation of human dental pulp stem
cells in vitro, Cell Biol Int 2008; 32: 827-834; Robey P G, Stem
cells near the century mark, J Clin Invest 2000; 105:
1489-1491).
SUMMARY OF INVENTION
Problem to be Solved by Invention
[0015] Nonetheless, it is still unclear how dental pulp stem cells
such as SHED or DPSCs can be medically applied, and specific target
diseases thereof are not known at all.
[0016] An object of the present invention is to provide a novel
therapeutic means that utilizes dental pulp stem cells.
Means for Solving the Problem
[0017] The present invention encompasses the following aspects:
[0018] [1] A damaged part treatment composition for repairing a
damaged part of a target tissue, the composition including a stem
cell-conditioned medium obtained by culturing stem cells.
[0019] [2] The damaged part treatment composition according to [1],
which does not include the stem cells.
[0020] [3] The damaged part treatment composition according to [1]
or [2], wherein the stem cell-conditioned medium includes at least
two cytokines.
[0021] [4] The damaged part treatment composition according to any
one of [1] to [3], wherein the stem cell-conditioned medium
includes at least two cytokines selected from the group consisting
of vascular endothelial growth factor (VEGF), hepatocyte growth
factor (HGF), insulin-like growth factor (IGF), platelet-derived
growth factor (PDGF) and transforming growth factor .beta.
(TGF-.beta.).
[0022] [5] The damaged part treatment composition according to any
one of [1] to [4], wherein the stem cells are somatic stem
cells.
[0023] [6] The damaged part treatment composition according to any
one of [1] to [5], wherein the stem cells are derived from
mesenchymal stem cells.
[0024] [7] The damaged part treatment composition according to any
one of [1] to [6], wherein the stem cells are dental pulp stem
cells.
[0025] [8] The damaged part treatment composition according to any
one of [1] to [7], which does not include any serum.
[0026] [9] The damaged part treatment composition according to any
one of [1] to [8], wherein the treatment of a damaged part includes
treatment of damage to skin, periodontal tissue or bone, treatment
of cerebral infarction or treatment of a central nervous system
(CNS) disease.
[0027] [10] The damaged part treatment composition according to any
one of [1] to [9], wherein the treatment of a damaged part includes
treatment of a CNS disease, and the CNS disease is a disease or
disorder selected from the group consisting of a spinal cord
injury, a neurodegenerative disorder, degeneration or loss of
neuronal cells and a retinal disease involving a neuronal cell
disorder.
[0028] [11] A method of producing the damaged part treatment
composition of any one of [1] to [10], the method including the
following steps (1) to (3):
[0029] (1) a step of selecting adhesive cells from dental pulp
cells;
[0030] (2) a step of culturing the adhesive cells; and
[0031] (3) a step of collecting a conditioned medium.
[0032] [12] The production method according to [11], further
including the following step (4):
[0033] (4) a step of subjecting the collected conditioned medium to
at least one treatment selected from the group consisting of
centrifugation, concentration, solvent substitution, dialysis,
freezing, drying, freeze-drying, dilution, desalting and
storage.
[0034] [13] The production method according to [11] or [12],
further including one of the following steps (a) or (b):
[0035] (a) a step of checking the collected conditioned medium with
respect to the presence or absence of a neurite outgrowth activity
in the presence of a nerve regeneration inhibitory substance;
or
[0036] (b) a step of checking the collected conditioned medium with
respect to the presence or absence of an apoptosis inhibitory
activity toward neuronal cells.
[0037] [14] A damaged part treatment method for repairing a damaged
part of a target tissue, the method including administering the
damaged part treatment composition of any one of [1] to [10] to a
patient having the target tissue for the damaged part treatment
composition, in an amount effective for repairing the damaged part
of the target tissue.
[0038] [15] The damaged part treatment method according to [14],
wherein the administrating is such that the repairing of the
damaged part is achieved based on an ability of endogenous stem
cells.
[0039] [16] The damaged part treatment method according to [14] or
[15], wherein the damaged part treatment composition is
administered by an administration method selected from the group
consisting of intravenous administration, intraarterial
administration, intraportal administration, intradermal
administration, subcutaneous administration, intramuscular
administration, intraperitoneal administration and intranasal
administration.
[0040] [17] A method of treating cerebral infarction including
administering the damaged part treatment composition of any one of
[1] to [10] to a cerebral infarction patient, in an amount
therapeutically effective for repairing a damaged part of the
brain.
[0041] [18] The method of treating cerebral infarction according to
[17], wherein the damaged part treatment composition is
administered by intranasal administration.
[0042] [19] A method of treating a CNS disease including
administering the damaged part treatment composition of any one of
[1] to [10] as a CNS disease treatment composition to a CNS disease
patient, in a therapeutically effective amount.
[0043] [20] The treatment method according to [19], wherein a
dental pulp stem cell is administered to the CNS disease patient
simultaneously with, or after, the administering of the CNS disease
treatment composition.
[0044] [21] The treatment method according to [20], wherein the
dental pulp stem cell is an undifferentiated dental pulp stem cell
that has not been subjected to differentiation-inducing treatment
after obtainment thereof, or a differentiation-induced dental pulp
stem cell that has been induced to differentiate into a neural cell
after obtainment thereof.
[0045] [22] The treatment method according to any one of [19] to
[21], wherein a pluripotent stem cell that has been induced to
differentiate into a neural cell is administered to the CNS disease
patient after the administering of the CNS disease treatment
composition.
[0046] [23] A method of determining whether or not a prepared
dental pulp stem cell-conditioned medium is effective as an active
ingredient of the CNS disease treatment composition to be employed
in the CNS disease treatment method according to any one of claims
19 to 22, the method including at least one of the following steps
(a) or (b):
[0047] (a) a step of checking the conditioned medium with respect
to the presence or absence of a neurite outgrowth activity in the
presence of a nerve regeneration inhibitory substance; or
[0048] (b) a step of checking the conditioned medium with respect
to the presence or absence of an apoptosis inhibitory activity
toward neuronal cells.
Advantageous Effect of Invention
[0049] According to the invention, a novel treatment means
utilizing a dental pulp stem cell, specifically a damaged part
treatment composition and a production method thereof, and a
damaged part treatment method using the damaged part treatment
composition, can be provided.
BRIEF DESCRIPTION OF DRAWINGS
[0050] FIG. 1 is a diagram illustrating an experimental design
using hairless mice. Wrinkle was induced by UVB irradiation.
[0051] FIG. 2 is a view showing the morphology, immunological
analysis and proliferation rates of various types of cells. (A) to
(C) respectively represent (A) BMSC, (B) DPSC and (C) SHED
(.times.40). (D) to (F) represent immunofluorescence staining
images of the stem cell marker STRO-1. (D) BMSC, (E) DPSC and (F)
SHED were positive for STRO-1 (green fluorescence). DAPI was used
to visualize the nuclei (blue fluorescence). (G) The proliferation
rates of SHED, DPSCs and BMSCs were assessed using BrdU. Bar:
standard deviation. Significance: *P<0.05.
[0052] FIG. 3 is a view showing evaluation of wrinkles by replica
analysis after SH-CM injection. (A) a group to be treated, (B) a
group treated with 100% SH-CM.
[0053] FIG. 4 is a diagram demonstrating amelioration of wrinkles
in natural level of SH-CM- and SHED-injected group.
[0054] FIG. 5 is a view showing Hematoxylin-Eosin staining images.
(A) SH-CM-treated group. (B) SHED-injected group.
[0055] FIG. 6 is a graph comparing the dermal thicknesses.
[0056] FIG. 7 is a diagram showing an effect of SH-CM on the
proliferation of HDF.
[0057] FIG. 8 shows a western blotting analysis demonstrating an
effect of SH-CM on collagen type I and MMP-1.
[0058] FIG. 9 is a conceptual diagram illustrating the mechanism of
bone regeneration using the composition of the invention.
[0059] FIG. 10 is an explanatory diagram illustrating the
experimental method according to Example 3 of the invention.
[0060] FIG. 11 is a view explaining the calculation of the BIC
value employed in Example 3 of the invention.
[0061] FIG. 12 shows staining images obtained as a result of
Example 3 of the invention.
[0062] FIG. 13 is a diagram showing the results of Example 3 of the
invention.
[0063] FIG. 14 is a photograph demonstrating the results of Example
3 of the invention.
[0064] FIG. 15 is an X-ray photograph showing the results of the
clinical case of Example 3 of the invention.
[0065] FIG. 16 is a view explaining the experimental model of
Example 4 of the invention.
[0066] FIG. 17 is a photograph explaining the treatment modalities
employed in Example 4 of the invention.
[0067] FIG. 18 is a photograph explaining the treatment modalities
employed in Example 4 of the invention.
[0068] FIG. 19 is a diagram explaining the treatment modalities
employed in Example 4 of the invention.
[0069] FIG. 20 shows staining images showing the regeneration state
of the cementum obtained as a result of Example 4 of the invention.
The upper photographs show the case of using GF, and the lower
photographs show the case of using PRP.
[0070] FIG. 21 is a graph showing the results of Example 4 of the
invention (N.sub.2--NC).
[0071] FIG. 22 is a graph showing the results of Example 4 of the
invention (N.sub.1-JE).
[0072] FIG. 23 is a photograph showing the pretreatment in the
clinical case of Example 4 of the invention.
[0073] FIG. 24 is a photograph explaining the manner of treatment
in the clinical case of Example 4 of the invention.
[0074] FIG. 25 is a photograph showing the results of the clinical
case of Example 4 of the invention.
[0075] FIG. 26 is a diagram illustrating the induction of cerebral
infarction according to Example 5 of the invention.
[0076] FIG. 27 is a graph showing changes in disability score after
starting the nasal administration to Group I, Group II and Group
III in Example 5 of the invention.
[0077] FIG. 28 is a graph showing the infarct volumes on day 16
after starting the nasal administration to Group I, Group II and
Group III in Example 5 of the invention.
[0078] FIG. 29 is a conceptual diagram explaining a preparation
method of the conditioned medium. hSHED: stem cell from human
exfoliated deciduous teeth. hDPSC: permanent teeth dental pulp stem
cell. hBMSC: human bone marrow mesenchymal stem cell. hFibroblast:
human fibroblast.
[0079] FIG. 30 is a photograph showing the results of a neurite
outgrowth experiment (phase-contrast microscopic image).
[0080] FIG. 31 shows the results of a neurite outgrowth experiment.
The graph shows the proportion of cells of which neurites were
observed (left) and neurite length (right).
[0081] FIG. 32 is a photograph showing the results of a neurite
outgrowth experiment (phase-contrast microscopic image).
[0082] FIG. 33 shows the result of a neurite outgrowth experiment.
The graph shows the proportion of cells of which neurites were
observed (left) and neurite length (right).
[0083] FIG. 34 is a photograph showing the results of an apoptosis
inhibition experiment (TUNEL assay).
[0084] FIG. 35 shows the result of an apoptosis inhibition
experiment (TUNEL assay). The graph shows statistically-processed
apoptosis inhibiting effects of the conditioned medium from dental
pulp stem cells. The left graph is a graph in which apoptosis
inhibiting effects in the presence of CSPG are checked, and the
right graph is a graph in which apoptosis inhibiting effects in the
presence of MAG are checked.
[0085] FIG. 36 is a graph showing the results of an experiment
using an animal model of spinal cord crush injury. SHED-CM: dental
pulp stem cell-conditioned medium administered group. BMSC-CM: bone
marrow mesenchymal stem cell-conditioned medium administered group.
Control: PBS administered group.
[0086] FIG. 37 shows the results of an experiment using an animal
model of spinal cord crush injury. Comparison between the control
group and the SHED-CM group is shown in terms of the bone marrow
state (upper photograph) and the spinal cord weight (lower
graph).
[0087] FIG. 38 shows the results of an experiment using an animal
model of spinal cord crush injury. SHED-CM: dental pulp stem
cell-conditioned medium administered group. Control: PBS
administered group.
[0088] FIG. 39 shows the results of an experiment using an animal
model of spinal cord crush injury. SHED-CM: dental pulp stem
cell-conditioned medium administered group. Control: PBS
administered group.
BEST EMBODIMENT FOR CARRYING OUT THE INVENTION
Damaged Part Treatment Composition
[0089] The damaged part treatment composition of the invention is a
damaged part treatment composition for repairing a damaged part of
a target tissue, the composition including a stem cell-conditioned
medium obtained by culturing stem cells.
[0090] The damaged part treatment method of the invention is a
damaged part treatment method for repairing or restoring a damaged
part of a target tissue, the method including administering the
damaged part treatment composition to a patient having the target
tissue for the damaged part treatment composition, in an amount
effective for repairing the damaged part of the target tissue.
[0091] When the application of SHED to wound healing was still an
inference, the inventors of the invention studied the relationship
between a growth factor derived from stem cells from human
exfoliated deciduous teeth (SHED) and human dermal fibroblasts
(HDFs) for the first time. SHED affect HDFs by enhancing collagen
synthesis and activating the growth and migration activity of HDFs.
This suggests that SHED or SHED-derived conditioned medium (SH-CM)
can be used for treatment of photo-aging. The results suggest that
SHED and SH-CM should be structurally suitable for treatment of
photo-aging. SHED contribute to enhancement of the wound healing
activity of HDFs, mainly with a secreted growth factor or
extracellular matrix protein. Further study of the mechanism using
neutralizing antibodies against respective growth factors clarifies
the roles of soluble factors from SHED in the process of wound
healing. Further, deciduous teeth naturally exfoliate during
infancy, and are usually disposed of as they are. Therefore,
utilization of stem cells from human exfoliated deciduous teeth has
a great advantage in terms of the absence of invasiveness of the
obtainment thereof and morality problem for utilization.
[0092] According to the invention, a stem cell-conditioned medium
obtained by culturing stem cells is used as an active ingredient
for a damaged part treatment composition. When the stem
cell-conditioned medium, which contains a cytokine mixture, is
applied to a damaged part, the stem cell-conditioned medium induces
cell growth in the damaged part, as a result of which the tissue
having the damaged part can be repaired. In an embodiment of the
invention, it can be presumed that the mixture of cytokines in the
stem cell-conditioned medium used in the invention serves as an
inductive signal for endogenous stem cells in the target tissue,
and, therefore, the endogenous stem cells can differentiate and
proliferate. As a result, the proliferation of cells, generation of
extracellular matrix, etc. may occur in the damaged part of the
target tissue. From these, it is thought that a tissue having a
damaged part can be repaired based on such regenerative ability of
endogenous stem cells in the target tissue.
[0093] For example, secretion of several growth factors involved in
skin regeneration from MSCs allows SHED and SHED-derived growth
factor to reverse UVB-induced photo-damage. Therefore, wrinkles
were induced in hairless mice by an eight-week regimen of UVB
irradiation, and an anti-wrinkle effect by the subcutaneous
injection of SHED and its conditioned medium was investigated. In
addition, mechanisms for improving wrinkling via paracrine routes
by further using SH-CM in cultured HDFs were investigated.
[0094] In the invention, the term "damaged part" means a part in a
tissue that became unable to perform its original function due to
occurrence of physical or physiological defect in the tissue, and
the concept thereof encompasses external injury as well as a
injured part, dysfunctional part or diseased part caused by
physical or physiological defect of the tissue.
[0095] In the invention, "repair" means that some or all of the
functions that was lost due to damage to the target tissue are
maintained or recovered as compared to the functions of the damaged
part at the time of damaging, and broadly encompasses recovery of
the functions of the tissue as well as regeneration as a functional
tissue. The assessment for the maintenance or recovery of the
functions may be carried out based on, for example, an assay
usually employed for the assessment of the appearance and the
degree of the function of interest, although the assessment varies
depending on the damaged tissue.
[0096] Examples of somatic stem cells used in the invention
include, but are not limited to, stem cells from the dermal system,
the digestive system, the bone marrow system, the nervous system,
etc. Examples of the somatic stem cells in the dermal system
include epidermal stem cells, hair follicle stem cells, etc.
Examples of the somatic cells in the digestive system include
pancreatic (common) stem cells, hepatic stem cells, etc. Examples
of the somatic cells in the bone marrow system include
hematopoietic stem cells, mesenchymal stem cells, etc. Examples of
somatic stem cells in the nervous system include neural stem cells,
retinal stem cells, etc. Somatic cells used in the invention may be
naturally-occurring or genetically-modified as long as they can
achieve the intended treatment.
[0097] The origins of stem cells are classified into ectoderm,
endoderm and mesoderm. Stem cells of ectodermal origin are present
mainly in the brain, and include neural stem cells. Stem cells of
endodermal origin are present mainly in the bone marrow, and
include blood vessel stem cells, hematopoietic stem cells,
mesenchymal stem cells, etc. Stem cells of mesoderm origin are
present mainly in organs, and include hepatic stem cells,
pancreatic stem cells, etc.
[0098] In the invention, it is preferable to use somatic stem cells
which may be derived from any mesenchyme, more preferably somatic
stem cells derived from dental pulp, and most preferably somatic
stem cells derived from human exfoliated deciduous teeth. Somatic
stem cells from mesenchyme may produce various cytokines such as
vascular endothelial growth factor (VEGF), hepatocyte growth factor
(HGF), insulin-like growth factor (IGF), platelet-derived growth
factor (PDGF), transforming growth factor-.beta. (TGF-.beta.)-1 and
-3, TGF-.alpha., KGF, HBEGF and SPARC. In the invention, the stem
cell-conditioned medium preferably includes at least two cytokines,
and more preferably includes a combination of two or more selected
from the group consisting of vascular endothelial growth factor
(VEGF), hepatocyte growth factor (HGF), insulin-like growth factor
(IGF), platelet-derived growth factor (PDGF) and transforming
growth factor .beta. (TGF-.beta.).
[0099] The mixture of cytokines for use in the invention may be
used as a part of the stem cell-conditioned medium or as a mixture
of cytokines that has been isolated from the stem cell-conditioned
medium. In the mixture of cytokines isolated from the stem
cell-conditioned medium, a part of the cytokines may be replaced
with one or more known corresponding cytokine.
[0100] The stem cell-conditioned medium for use in the invention is
preferably obtained from a culture of somatic stem cells derived
from the same individual as that having the target tissue, in order
to avoid rejection. The target tissue may be the same as or
different from a tissue from which the somatic stem cell used to
obtain the stem cell-conditioned medium is derived.
[0101] A stem cell-conditioned medium used in the invention is not
limited to a stem cell-conditioned medium obtained from culturing
somatic stem cells, and may contain a stem cell-conditioned medium
obtained from culturing embryonic stem cells (ES cells), induced
pluripotent stem cells (iPS cells), embryonal carcinoma cells (EC
cells) or the like.
[0102] The somatic stem cell-conditioned medium is a medium
obtained by culturing somatic stem cells, and does not include the
cells themselves. The conditioned medium that can be used in the
invention can be obtained by, for example, removing cell components
by separation after culturing. The conditioned medium may be
subjected to various treatments (such as centrifugation,
concentration, solvent substitution, dialysis, freezing, drying,
freeze-drying, dilution, desalting or storage), as appropriate,
before use.
[0103] The stem cells for obtaining the stem cell-conditioned
medium can be selected by an ordinary method, and can be selected
based on the size and morphology of cells, or as adhesive cells. In
the case of dental pulp stem cells, the stem cells can be selected
as adhesive cells from dental pulp cells obtained from exfoliated
deciduous teeth or permanent teeth, or as subcultured cells
thereof, as described below. The later-described method of
producing a CNS disease treatment composition can preferably be
used as a method of producing the damaged part treatment
composition. The dental pulp stem cell-conditioned medium to be
used may be a conditioned medium obtained by culturing the selected
stem cells. In the case of using other stem cells, the stem
cell-conditioned medium can be obtained after obtaining target stem
cells from a tissue that may contain the target stem cells in a
similar manner.
[0104] The "stem cell-conditioned medium" is defined as a medium
that is obtained by culturing stem cells, and that does not include
cells themselves. The composition of the invention includes the
"stem cell-conditioned medium" as an active ingredient. In an
aspect of the composition, the composition as a whole does not
include any cells (regardless of the type of cells). The
composition according to this aspect is clearly distinguished from
the stem cells themselves as a matter of course, and from various
compositions that contain stem cells, due to the feature described
above. A typical example of this aspect is a composition that does
not include any stem cells, and that consists only of the stem
cell-conditioned medium.
[0105] A basal medium, or a medium obtained by adding serum or the
like to a basal medium, can be used for the stem cell culture
medium. In the case of preparing a serum-free "dental pulp stem
cell-conditioned medium", it is preferable to use a serum-free
medium throughout the entire process or to use a serum-free medium
at subculturing for the last passage, or for the last few passages.
DMEM, Iscove's Modified Dulbecco's Medium (IMDM) (GIBCO
Corporation, etc.), Ham's F12 medium (HamF12) (Sigma-Aldrich
Corporation, GIBCO Corporation, etc.), RPMI1640 medium, etc., can
be used as the basal medium. Two or more basal media may be used in
combination. An example of a mixed medium is a medium formed by
mixing equivalent amounts of IMDM and HamF12 (commercially
available as, for example, IMDM/HamF12 (tradename, GIBCO
Corporation)). Examples of ingredients that can be added to the
medium include serums (such as fetal bovine serum, human serum and
sheep serum), serum replacements (knockout serum replacement (KSR),
etc.), bovine serum albumin (BSA), antibiotics, various vitamins
and various minerals.
[0106] For the cultivation of stem cells, usually-employed
conditions can be applied as they are. The method for producing a
stem cell-conditioned medium may be the same as the later-described
method of producing a CNS disease treatment composition, except for
appropriately modifying the step of isolation and selection of stem
cells in accordance with the type of stem cells. Those skilled in
the art would be able to appropriately carry out the isolation and
selection of stem cells in accordance with the type of stem
cells.
[0107] The target tissue in the invention is not particularly
limited, and examples thereof include skin, bone, periodontal
tissue, brain, etc. The composition of the invention is effective
for repairing such target tissues. As an example, FIG. 9 shows a
conceptual diagram of the mechanism of bone regeneration using the
composition of the invention.
[0108] The composition of the invention is also effective for the
treatment of disorders related to tissue damage. Examples of such
disorders include cerebral infarction, periodontal disease, spinal
cord injury, skin ulceration, osteoporosis, etc. In other words,
the composition of the invention is a composition for treatment of
cerebral infarction, periodontal disease, spinal cord injury, skin
ulceration, osteoporosis, etc., and includes a stem
cell-conditioned medium obtained by culturing somatic stem cells.
For example, the damaged part treatment composition of the
invention is used as a composition for treatment of a damaged part,
such as treatment of a damage to skin, periodontal tissue or bone,
treatment of cerebral infarction or treatment of CNS disease. The
dosage of the damaged part treatment composition may be any
therapeutically effective amount. When the damaged part treatment
composition, which includes the stem cell-conditioned medium as an
active ingredient, is used for treatment, the dosage of the damaged
part treatment composition may be adjusted, as appropriate. The
damaged part treatment composition may be used after concentrating
the active ingredient as described below.
[0109] Other ingredients may additionally be used in the
composition of the invention in accordance with the state of the
subject to which the composition is applied, as long as the
expected therapeutic effect is maintained. Some examples of
ingredients that can additionally be used in the invention include
the following:
[0110] (i) Bioabsorbable Materials
[0111] Hyaluronic acid, collagen, fibrinogen (for example, BOLHEAL
(registered trademark)), etc., may be used as organic bioabsorbable
materials.
[0112] (ii) Gelling Materials
[0113] Gelling materials for use preferably have high bioaffinity,
and hyaluronic acid, collagen, fibrin adhesive or the like may be
used. Various hyaluronic acids and collagens may be selected and
used, and it is preferable to adopt those suitable for the purpose
of application of the composition of the invention (the tissue to
which the composition is to be applied). Collagens to be used are
preferably soluble (acid-soluble collagens, alkali-soluble
collagens, enzyme-solubilized collagens, etc.).
[0114] (iii) Others
[0115] Other pharmaceutically-acceptable ingredients (for example,
carriers, excipients, disintegrants, buffer agents, emulsifying
agents, suspending agents, soothing agents, stabilizers,
preservatives, antiseptic agents, physiological saline, etc.) may
be contained. Lactose, starch, sorbitol, D-mannitol, white sugar,
etc. may be used as excipients. Starch, carboxymethylcellulose,
calcium carbonate, etc. may be used as disintegrants. Phosphoric
acid salts, citric acid salts, acetic acid salts, etc. may be used
as buffering agents. Gum arabic, sodium alginate, Tragacanth, etc.
may be used as emulsifying agents. Glycerin monostearate, aluminum
monostearate, methylcellulose, carboxymethylcellulose,
hydroxymethylcellulose, sodium lauryl sulfate, etc. may be used as
suspending agents. Benzyl alcohol, chlorobutanol, sorbitol, etc.
may be used as soothing agents. Propyleneglycol, ascorbic acid,
etc. may be used as stabilizers. Phenol, benzalkonium chloride,
benzylalcohol, chlorobutanol, methylparaben, etc. may be used as
preservatives. Benzalkonium chloride, parahydroxybenzoic acid,
chlorobutanol, etc. may be used as antiseptic agents. Antibiotics,
pH adjusting agents, growth factors (such as epidermal growth
factor (EGF), nerve growth factor (NGF) and brain-derived
neurotrophic factor (BDNF)), etc. may also be contained.
[0116] The final form of the composition of the invention is not
particularly limited. Examples of the form include liquid forms
(such as a purely liquid form and a gel form), and solid forms
(such as a powdery form, a fine grain form and a granular
form).
[0117] Other aspects of the invention include a method of repairing
a damaged part of a target tissue and a method of treating a
damaged tissue. These methods include administering the stem
cell-conditioned medium to the damaged part of the target tissue or
the damaged tissue. Due to the administering, the target tissue
having the damaged part can effectively be repaired. In particular,
in a case in which the target tissue is brain, the methods can
preferably applied as a method of treating cerebral infarction.
[0118] The method and route of the administration of the damaged
part treatment composition are not particularly limited. For
example, the damaged part treatment composition is preferably
administered parenterally, and the parenteral administration may be
systemic administration or topical administration. Examples of
topical administration include injection, application or spraying
to the target tissue, etc. Examples of the method of administering
the damaged part treatment composition include intravenous
administration, intraarterial administration, intraportal
administration, intradermal administration, subcutaneous
administration, intramuscular administration, intraperitoneal
administration, intranasal administration, etc. In particular,
intranasal administration or the like is preferable due to its low
invasiveness. The dosage regimen may be, for example, from once to
several times a day, once every two days, once every three days, or
the like. The dosage regimen may be prepared in consideration of
the sex, age, weight, pathological condition, etc. of the subject
(recipient).
[0119] The selection of the administration method may be carried
out by a person skilled in the art, based on the type of target
tissue, the type of disease to be treated, etc. For example,
application of intranasal administration or the like is
particularly preferable for, for example, the treatment of a
disorder or repair of a damaged tissue of which target tissue is
located in the brain, because the intranasal administration is less
invasive and free from the need to consider the passage through the
blood-brain barrier. For example, intranasal administration may
preferably be applied in a case in which the target tissue is
brain. Intranasal administration may preferably be applied to
treatment of cerebral infarction.
[0120] The subject to which the damaged part treatment composition
is administered is typically a human patient having damage in the
target tissue. However, application to mammals other than human
(including pet animals, farm animals and laboratory animals,
specific examples of which include mice, rats, guinea pigs,
hamsters, monkeys, cattle, pigs, goats, sheep, dogs, cats, etc.) is
also contemplated.
[0121] The method of treating cerebral infarction of the invention
includes intranasally administering the stem cell-conditioned
medium, to repair a damaged part of the brain. According to this
treatment method, a region that was damaged by cerebral infarction
can effectively be restored with less invasiveness.
[0122] <CNS Disease Treatment Composition>
[0123] Other aspects of the invention encompass, particularly, a
CNS disease treatment composition and a method of treating a CNS
disease.
[0124] The inventors carried out research under the circumstance
discussed above. The inventors have clarified that dental pulp stem
cells are a unique group of cells that coexpress all neural lineage
markers including neural stem cell markers, differentiated neural
cell markers, astrocyte markers and oligodendrocyte markers, and
that dental pulp stem cells highly express brain-derived
neurotrophic factor (BDNF), and have also demonstrated, through
animal experiments, that dental pulp stem cells induce nerve
regeneration (see Japanese Patent Application No. 2010-92585).
[0125] As described above, the inventors thus far looked for the
potential capacity of dental pulp stem cells (SHED, DPSCs), and
studied the utility thereof as cells from various viewpoints.
During the course of further advancing the research, the inventors
have drastically changed their viewpoint, and carried out various
experiments focusing on a dental pulp stem cell-conditioned medium.
Here, there is an already known fact that peripheral nerves easily
regenerate after being damaged, but central nerves (brain, spinal
cord) rarely regenerate. The biggest reason why the central nerve
regeneration does not occur is the presence of various factors that
inhibit outgrowth of regenerated axons in the CNS after being
damaged. Activated astrocyte-derived chondroitin sulfate
proteoglycan (CSPG), myelin-associated glycoprotein (MAG), etc.
have thus far been identified as nerve regeneration inhibitory
factors. These inhibitory substances inhibit neuronal axon
outgrowth via activation of intracellular protein Rho or ROCK, and
induce apoptosis. No agent has been found which inhibits apoptosis
even in the presence of nerve regeneration inhibitory factor, and
which exerts axon elongation effect. Analysis by the inventors
revealed a surprising fact that a dental pulp stem cell-conditioned
medium inhibits the action of nerve regeneration inhibitory
substances (cancels the inhibition), promotes outgrowth of
neurites, and suppresses apoptosis even in the environment of a
damaged CNS (i.e., the environment in which substances that inhibit
outgrowth of neurites and induce apoptosis are present). The
inventors further studied the activity of the dental pulp stem
cell-conditioned medium using model animals with injured spinal
cord, as a result of which the administration of the dental pulp
stem cell-conditioned medium remarkably improved the motor function
of hindlimbs. Further, as a result of histological evaluation, the
administration of the dental pulp stem cell-conditioned medium
suppressed morphological alteration of the spinal cord and
enlargement of nerve injury. As discussed above, excellent
regenerative and therapeutic effects of the dental pulp stem
cell-conditioned medium were confirmed also by animal
experiments.
[0126] As described above, earnest study by the inventors of the
invention resulted in a finding that the dental pulp stem
cell-conditioned medium is quite effective for regeneration and
healing of the CNS. The invention as discussed below is mainly
based on this finding. Here, the dental pulp stem cell-conditioned
medium is more advantageous than a case in which dental pulp stem
cells themselves are used, in terms of advance preparation and
storage, and the dental pulp stem cell-conditioned medium is
particularly suitable for the treatment of the acute or subacute
phase of CNS diseases. The utility of the dental pulp stem
cell-conditioned medium is quite high also in the sense that the
dental pulp stem cell-conditioned medium does not include any
cellular components and is capable of overcoming the immune
rejection problem.
[0127] The present aspect of the invention includes the
following:
[0128] [1] A CNS disease treatment composition including a dental
pulp stem cell-conditioned medium.
[0129] [2] The CNS disease treatment composition according to [1],
which exhibits a neurite outgrowth activity in the presence of a
nerve regeneration inhibitory substance.
[0130] [3] The CNS disease treatment composition according to [2],
wherein the nerve regeneration inhibitory substance is chondroitin
sulfate proteoglycan or myelin-associated glycoprotein.
[0131] [4] The CNS disease treatment composition according to any
one of [1] to [3], which exhibits an apoptosis inhibitory activity
toward neuronal cells.
[0132] [5] The CNS disease treatment composition according to any
one of [1] to [4], which does not include any dental pulp stem
cells.
[0133] [6] The CNS disease treatment composition according to any
one of [1] to [4], which is combined with dental pulp stem
cells.
[0134] [7] The CNS disease treatment composition according to [6],
wherein the dental pulp stem cells are undifferentiated dental pulp
stem cells that have not been subjected to differentiation-inducing
treatment after obtainment thereof [8] The CNS disease treatment
composition according to any one of [1] to [7], which does not
include serum.
[0135] [9] The CNS disease treatment composition according to any
one of [1] to [9], wherein the conditioned medium is a conditioned
medium obtained by culturing adhesive cells in dental pulp cells or
subcultured cells thereof
[0136] [10] The CNS disease treatment composition according to any
one of [1] to [9], wherein the CNS disease is a disease or disorder
selected from the group consisting of neurodegenerative diseases
such as spinal cord injury, amyotrophic lateral sclerosis,
Alzheimer's disease, Parkinson's disease, progressive supranuclear
palsy, Huntington's disease, multiple system atrophy and
spinocerebellar ataxia, degeneration or loss of neuronal cells
caused by cerebral ischemia, intracerebral hemorrhage or cerebral
infarction and a retinal disease involving a neuronal cell
disorder.
[0137] [11] A method of producing a CNS disease treatment
composition including the following steps (1) to (3):
[0138] (1) a step of selecting adhesive cells from dental pulp
cells;
[0139] (2) a step of culturing the adhesive cells; and
[0140] (3) a step of collecting a conditioned medium.
[0141] [12] The production method according to [11], wherein the
step (2) is carried out using a serum-free medium.
[0142] [13] The production method according to [11] or [12],
wherein the conditioned medium after subculturing is collected in
step (3).
[0143] [14] The production method according to any one of [11] to
[13], further including the following step (4):
[0144] (4) a step of subjecting the collected conditioned medium to
at least one treatment selected from the group consisting of
centrifugation, concentration, solvent substitution, dialysis,
freezing, drying, freeze-drying, dilution, desalting and
storage.
[0145] [15] The production method according to any one of [11] to
[14], further including the following step (a) and/or (b):
[0146] (a) a step of checking the collected conditioned medium with
respect to the presence or absence of a neurite outgrowth activity
in the presence of a nerve regeneration inhibitory substance;
and
[0147] (b) a step of checking the collected conditioned medium with
respect to the presence or absence of an apoptosis inhibitory
activity toward neuronal cells.
[0148] [16] The production method according to any one of [11] to
[15], wherein the CNS disease is a disease or disorder selected
from the group consisting of spinal cord injury, neurodegenerative
diseases such as amyotrophic lateral sclerosis, Alzheimer's
disease, Parkinson's disease, progressive supranuclear palsy,
Huntington's disease, multiple system atrophy and spinocerebellar
ataxia, degeneration or loss of neuronal cells caused by cerebral
ischemia, intracerebral hemorrhage or cerebral infarction and a
retinal disease involving a neuronal cell disorder.
[0149] [17] A method of treating a CNS disease including a step of
administering the CNS disease treatment composition of any one of
[1] to [10] to a CNS disease patient, in a therapeutically
effective amount.
[0150] [18] The treatment method according to [17], wherein a
dental pulp stem cell is administered to the CNS disease patient
simultaneously with, or after, the administering of the CNS disease
treatment composition.
[0151] [19] The treatment method according to [18], wherein the
dental pulp stem cell is an undifferentiated dental pulp stem cell
that has not been subjected to differentiation-inducing treatment
after obtainment thereof, or a differentiation-induced dental pulp
stem cell that has been induced to differentiate into a neural cell
after obtainment thereof
[0152] [20] The treatment method according to [17], wherein a
pluripotent stem cell that has been induced to differentiate into a
neural cell is administered to the CNS disease patient after the
administering of the CNS disease treatment composition.
[0153] [21] A method of determining whether or not a prepared
dental pulp stem cell-conditioned medium is effective as an active
ingredient of the CNS disease treatment composition, the method
comprising the following step (a) and/or (b):
[0154] (a) a step of checking the conditioned medium with respect
to the presence or absence of a neurite outgrowth activity in the
presence of a nerve regeneration inhibitory substance; and
[0155] (b) a step of checking the conditioned medium with respect
to the presence or absence of an apoptosis inhibitory activity
toward neuronal cells.
[0156] The CNS disease treatment composition of the invention
includes a dental pulp stem cell-conditioned medium. Dental pulp
stem cells are roughly classified into two types--dental pulp stem
cells from deciduous teeth and permanent teeth dental pulp stem
cells. In the present specification, dental pulp stem cells from
deciduous teeth are abbreviated to SHED, and permanent teeth dental
pulp stem cells are abbreviated to DPSCs, in accordance with
customary practices. Each of a SHED-conditioned medium and a
DPSC-conditioned medium can be used as a conditioned medium for
forming the CNS disease treatment composition.
[0157] The CNS disease treatment composition can be characterized
by the feature--"exhibiting a neurite outgrowth activity in the
presence of nerve regeneration inhibitory substance".
[0158] Dissimilar to the peripheral nervous system, nerve
regeneration inhibitory substances (neurite outgrowth inhibitory
factors) are present in the CNS. This is an important point when
CNS disease therapy (mainly nerve regeneration) is planned, and
needs consideration. Using the CNS disease treatment composition
having the feature described above allows for suppression of the
action of nerve regeneration inhibitory substances, and promotion
of nerve regeneration. Examples of the nerve regeneration
inhibitory substances are chondroitin sulfate proteoglycan (CSPG)
and myelin-associated glycoprotein (MAG). Whether or not the CNS
disease treatment composition has the feature can be confirmed by,
for example, an in vitro experimentation using neuronal cells and a
nerve regeneration inhibitory substance (CSPG or MAG) (see
later-described Examples with respect to the details of the
experimentation). A test composition is confirmed to have the
above-described feature if neurite outgrowth is observed when the
neuronal cells are cultured in the coexistence of the test
composition and CSPG or MAG
[0159] The CNS disease treatment composition can alternatively be
characterized by the feature--"exhibiting an apoptosis inhibiting
activity toward neuronal cells". Whether or not the CNS disease
treatment composition has this feature can be confirmed by, for
example, an in vitro experimentation using neuronal cells (see
later-described Examples with respect to the details of the
experimentation). A test composition is confirmed to have this
feature if cell death due to apoptosis is suppressed when the
neuronal cells are cultured in the presence of the test
composition. In a preferable aspect, the CNS disease treatment
composition has both of this feature and the above-described
feature (exhibiting a neurite outgrowth activity in the presence of
a nerve regeneration inhibitory substance).
[0160] In the present aspect, the term "dental pulp stem
cell-conditioned medium" refers to a medium that is obtained by
culturing dental pulp stem cells, and that does not include cell
components (i.e., dental pulp cells and dental pulp stem cells).
Therefore, a conditioned medium that can be used in the invention
can be obtained by, for example, removing cell components by
separation after culturing. The conditioned medium may be subjected
to various treatments (such as centrifugation, concentration,
solvent substitution, dialysis, freezing, drying, freeze-drying,
dilution, desalting or storage), as appropriate, before use.
Details of treatment methods for the conditioned medium are
described later.
[0161] Dental pulp stem cells can be selected as adhesive cells in
dental pulp cells. Therefore, a conditioned medium obtained by
culturing adhesive cells in dental pulp cells collected from
exfoliated deciduous teeth or permanent teeth, or subcultured cells
thereof, can be used as the dental pulp stem cell-conditioned
medium. Details of the method of preparing the dental pulp stem
cell-conditioned medium are described later.
[0162] As described above, the dental pulp stem cell-conditioned
medium is defined as a medium that is obtained by culturing dental
pulp stem cells and that does not include cell components. The CNS
disease treatment composition includes the dental pulp stem
cell-conditioned medium as an active ingredient, and, in one aspect
thereof, the composition as a whole does not include any cells
(regardless of the type of cells). The composition according to
this aspect is clearly distinguished from the dental pulp stem
cells themselves as a matter of course, and from various
compositions that contain dental pulp stem cells, based on the
feature described above. A typical example of this aspect is a
composition consisting only of the dental pulp stem
cell-conditioned medium.
[0163] One embodiment of the present aspect has characteristics in
that the dental pulp stem cell-conditioned medium and the dental
pulp stem cells are used in combination. Preferably, dental pulp
stem cells from deciduous teeth (SHED) are used in consideration of
their higher cell proliferation capacity compared to permanent
teeth dental pulp stem cells (DPSCs). Further, SHED are considered
to have higher differentiation capacity. A high BDNF expression
level of SHED (see, Japanese Patent Application No. 2010-92585),
which may provide higher therapeutic effects, is another advantage
of using SHED. In addition, SHED also has an advantage in that SHED
can be easily obtained.
[0164] In recent years, researches aiming to realize regenerative
medicine using cells have been carried out by many research groups.
In the case of using cells, cells obtained from a living body are
subjected to cultivation, selection, treatment or the like, and are
thereafter recovered and used as transplant components. In this
series of operations, a conditioned medium is usually disposed of
or replaced by, for example, physiological saline. Therefore, the
final transplant does not actively contain the conditioned medium.
In view of this, even the composition of the above-described
embodiment in which the dental pulp stem cell-conditioned medium
and the dental pulp stem cells are used in combination is literally
and practically distinguished from compositions or agents in which
the dental pulp stem cells are used as active ingredients with a
focus on the utility of dental pulp stem cells themselves, based on
the point that the composition of the above-described embodiment
includes the dental pulp stem cell-conditioned medium as an
essential active ingredient.
[0165] The embodiment described above is characterized by combined
use of the dental pulp stem cell-conditioned medium and the dental
pulp stem cells. The expression "combined use" as used herein means
that the dental pulp stem cell-conditioned medium and the dental
pulp stem cells are used together. Typically, the CNS disease
treatment composition is provided as a combination preparation in
which the dental pulp stem cell-conditioned medium and the dental
pulp stem cells are mixed. In such a case, it is preferable to use
dental pulp stem cells that have not been subjected to induction of
differentiation after obtainment thereof (i.e., dental pulp stem
cells that remain undifferentiated; also referred to as
"undifferentiated dental pulp stem cells" herein). In this case,
the CNS disease treatment composition exerts strong nerve
protection activity, and is thus suitable to, particularly,
application in the acute or subacute phase of CNS diseases (for
example, intractable neural diseases involving severe loss or
degeneration of neuronal cells, such as spinal cord injury or
cerebral infarction). The dental pulp stem cells used in this
embodiment are positive for the neural stem cell marker Nestin,
positive for the neural stem cell marker Doublecortin, positive for
the neuronal call marker .beta.-III tubulin, positive for the
neuronal call marker NeuN, positive for the astrocyte marker GFAP,
and positive for the oligodendrocyte marker CNPase, and highly
express BDNF (see, Japanese Patent Application No. 2010-92585).
[0166] The CNS disease treatment composition may also be provided
in the form of, for example, a kit composed of a first constituent
element containing the dental pulp stem cell-conditioned medium and
a second constituent element containing the dental pulp stem
cells.
[0167] In this case, to a subject to be treated (usually, a CNS
disease patient) administered with the first constituent element,
the second constituent element is administered simultaneously with
the administration of the first constituent element or after the
administration of the first constituent element. A regimen in which
the first constituent element and the second constituent element
are simultaneously administered is particularly suitable for
application in the acute or subacute phase of CNS diseases. In
order that high therapeutic effects are exerted in the case of
application in the acute or subacute phase, it is preferable to use
undifferentiated dental pulp stem cells as an active ingredient of
the second constituent element.
[0168] Here, the term "simultaneously" does not require exact
simultaneity. Accordingly, the concept of "simultaneously"
encompasses a case in which both elements are administered with no
time lag such as administration to the subject after mixing of both
constituent elements, as well as a case in which both constituent
elements are administered with substantially no time lag such as
administration of one of the constituent elements immediately after
the administration of the other one of the constituent
elements.
[0169] According to a regimen in which the first constituent
element is administered in the acute or subacute phase, and the
second constituent element is administered thereafter (for example,
3 days to 1 week after the administration of the first constituent
element), continuous and comprehensive therapeutic effects can be
expected. In a case in which this regimen is planned, it is
preferable to use dental pulp stem cells that have been induced to
differentiate into neural cells (here also referred to as
"differentiation-induced dental pulp stem cells") as an active
ingredient of the second constituent element. Here, the scope of
the term "neural cells" encompasses motor neurons,
dopamine-producing cells, various CNS cells, astrocytes,
oligodendrocytes and Schwann cells. The type of neural cells into
which the dental pulp stem cells are to be induced to differentiate
may be determined in consideration of the disease and pathological
condition of the subject to be treated. For example, for the
treatment of spinal cord injury, dental pulp stem cells that have
been induced to differentiate into mature nerve cells,
oligodendrocytes or Schwann cells may be used in the second
constituent element. An example of a method of inducing neural
differentiation is described below.
[0170] A method composed of the following two steps may be used for
induction of differentiation into dopamine-producing neuronal
cells. In the first step, dental pulp stem cells are cultured for 2
to 3 days in, for example, a DMED medium that contains 12.5 U/mL
Nystatin, N2 supplement, 20 ng/mL bFGF and 20 ng/mL EGF, using a
dish coated with poly-L-lysine. As a result of this step, neural
stem cell differentiation of the dental pulp stem cell is induced.
In the second step, the cells after the first step are cultured for
6 to 7 days in, for example, a Neurobasal.TM. medium that contains
B27 supplement, 1 mM db-cAMP, 0.5 mM IBMX, 200 .mu.M ascorbic acid
and 50 ng/mL BDNF. The induced dopamine-producing neuronal cells
can be confirmed by immunostaining using an anti-tyrosine
hydroxylase antibody. Besides the above method, various methods
that have been reported as methods for inducing differentiation of
neural stem cells or embryonic stem cells into dopamine-producing
neuronal cells, such as a method of culturing in the presence of
bFGF followed by floating culture of aggregates (Studer, L. et al.:
Nat. Neurosci., 1: 290-295, 1998), a method of culturing in the
presence of bFGF and glia cell-conditioned medium (Daadi, M. M. and
Weiss, S. J.: Neuroscience, 19: 4484-4497, 1999), a method
utilizing FGF8, Shh, bFGF, ascorbic acid, etc. (Lee, S. H. et al.:
Nat. Biotechnol., 18: 675-679, 2000), and a method of co-culturing
with bone marrow stromal cells (Kawasaki, H. et al.: Neuron, 28:
31-40, 2000), may be utilized after appropriate modification
thereof, if necessary.
[0171] A method composed of the following two steps may be used for
induction of astrocyte differentiation. In the first step, dental
pulp stem cells are cultured for four days in, for example, a
DMEM/F12 medium that contains N2 supplement and 10 ng/mL bFGF,
using a dish doubly coated with poly-L-ornithine and fibronectin.
In the second step, the cells are cultured for three days in the
medium further added with 80 ng/mL LIF and 80 ng/mL BMP2. The
differentiation induced astrocytes can be confirmed by
immunostaining using an anti-GFAP antibody.
[0172] A method composed of the following two steps may be used for
induction of oligodendrocyte differentiation. Similar to the
induction of astrocyte differentiation, in the first step, dental
pulp stem cells are cultured for four days in, for example, a
DMEM/F12 medium that contains N2 supplement, 10 ng/mL bFGF and 0.5%
FCS, using a dish doubly coated with poly-L-ornithine and
fibronectin. As a result of this step, the dental pulp stem cells
are induced into oligodendrocyte progenitor cells. In the
subsequent second step, the cells are cultured for four days in a
DMEM/F12 medium that contains 20 ng/mL T3 (Triiodothyronine), 20
ng/mL T4 (Thyroxine) and N2 supplement. The differentiation induced
oligodendrocytes can be confirmed using an anti-04 antibody.
[0173] As a component of the second constituent element,
pluripotent stem cells that have been induced to differentiate into
neural cells may be used in addition to, or in place of, the
differentiation induced dental pulp stem cells. Examples of
pluripotent stem cells include induced pluripotent stem cells (iPS
cells) and embryonic stem cells (ES cells). The "induced
pluripotent stem cells (iPS cells)" are cells having pluripotency
(multipotency), and proliferative capacity that are produced by
reprogramming somatic cells by introduction of reprogramming
factors. The induced pluripotent stem cells exhibit properties
similar to those of ES cells. The iPS cells can be produced by
various iPS cell production methods that have been reported thus
far. Of course, application of iPS cell production methods that
will be developed in the future is also contemplated, as a matter
of course.
[0174] The most basic technique among iPS cell production methods
is a method of introducing the four transcriptional factors of
Oct3/4, Sox2, KIF4 and c-Myc into a cell using a virus (Takahashi
K, Yamanaka S: Cell 126 (4), 663-676, 2006; Takahashi, K, et al:
Cell 131 (5), 861-72, 2007). Establishment of human iPS cells by
introducing the four factors of Oct4, Sox2, Lin28 and Nonog is also
reported (Yu J, et al: Science 318 (5858), 1917-1920, 2007).
Establishment of iPS cells by introducing the three factors other
than c-Myc (Nakagawa M, et al: Nat. Biotechnol. 26 (1), 101-106,
2008), the two factors of Oct3/4 and Klf4 (Kim J B, et al: Nature
454 (7204), 646-650, 2008) or only Oct3/4 (Kim J B, et al: Cell 136
(3), 411-419, 2009) has also been reported. Further, a technique of
introducing proteins as expression products of the genes into a
cell (Zhou H, Wu S, Joo J Y, et al: Cell Stem Cell 4, 381-384,
2009; Kim D, Kim C H, Moon J I, et al: Cell Stem Cell 4, 472-476,
2009) has also been reported. There is also a report that the use
of, for example, an inhibitor (BIX-01294) against histone
methyltransferase G9a, histone deacetylase inhibitor valproic acid
(VPA) or BayK8644 allows for improvement in the production
efficiency, reduction of factors to be introduced, etc (Huangfu D,
et al: Nat. Biotechnol. 26 (7), 795-797, 2008; Huangfu D, et al:
Nat. Biotechnol. 26 (11), 1269-1275, 2008; Silva J, et al: PLoS.
Biol. 6 (10), e 253, 2008). Studies on gene introduction methods
have also been carried out, and techniques using retroviruses, as
well as lentiviruses (Yu J, et al: Science 318(5858), 1917-1920,
2007), adenovirus (Stadtfeld M, et al: Science 322 (5903), 945-949,
2008), plasmids (Okita K, et al: Science 322 (5903), 949-953,
2008), transposon vectors (Woltjen K, Michael I P, Mohseni P, et
al: Nature 458, 766-770, 2009; Kaji K, Norrby K, Pac a A, et al:
Nature 458, 771-775, 2009; Yusa K, Rad R, Takeda J, et al: Nat
Methods 6, 363-369, 2009), or episomal vectors (Yu J, Hu K,
Smuga-Otto K, Tian S, et al: Science 324, 797-801, 2009) for
transfection have been developed.
[0175] Cells in which transformation into iPS cells, i.e.,
reprogramming, has occurred can be selected using, for example,
expression of a pluripotent stem cell marker (undifferentiated
marker) such as Fbox15, Nanog, Oct/4, Fgf-4, Esg-1 or Cript as an
indicator. The selected cells are collected as iPS cells.
[0176] Several types of ES cells are provided from public
institutions or commercially available. Examples of mouse ES cells
include ES-E14TG2a cells (ATCC), ES-D3 cells or the like (ATCC), H1
cells (Riken BioResource Center, Tsukuba-city, Japan), B6G-2 cells
(Riken BioResource Center, Tsukuba-city, Japan), R1 cells (Samuel
Lunenfeld Research Institute, Toronto, Canada), mouse ES cells
(129SV, catalogue number R-CMTI-1-15, R-CMTI-1A) (Dainippon
Sumitomo Pharma Co., Ltd., Osaka, Japan) and mouse ES cells
(C57/BL6, catalogue number R-CMTI-2A (Dainippon Sumitomo Pharma
Co., Ltd., Osaka, Japan). Monkey ES cells are available from, for
example, Stem Cell Research Center, Institute for Frontier Medical
Sciences, Kyoto University. Human ES cells are available from, for
example, Stem Cell Research Center, Institute for Frontier Medical
Sciences, Kyoto University, WiCell Research Institute (Madison,
USA), and ES Cell International Pte Ltd (Singapore). Methods for
establishing ES cells have been achieved, and part thereof has been
practice routinely. Therefore, one can himself establish desired ES
cells using ordinary methods. For example, Nagy. A. et al. eds.,
Manipulating the Mouse Embryo, A Laboratory Manual, Third Edition,
Cold spring Harbor Laboratory Press, 2003, Jikken-igaku Bessatsu
Baiyousaiboujikkenn Handbook (Culture Cell Experiment Handbook
(supplementary volume of Experimental medicine)), Yodosha Co., Ltd.
may be referenced with respect to method for establishing mouse ES
cells. For methods for establishing monkey ES cells, Suemori H,
Tada T, Torii R, et al., Dev Dyn 222, 273-279, 2001, etc. may be
referenced. For method for establishing human ES cells, Wassarman,
P. M. et al.: Methods in Enzymology, Vol. 365 (2003), etc. may be
referenced.
[0177] The CNS disease treatment composition is preferably free
from serum. The absence of serum in the CNS disease treatment
composition improves the safety of the composition. For example, a
serum-free conditioned medium can be prepared by culturing dental
pulp stem cells in a medium that does not contain any serum
(serum-free medium). In the case of subculturing for one passage or
plural passages, a serum-free conditioned medium can be obtained by
carrying out subculturing for the last passage, or for the last few
passages, in a serum-free medium. A serum-free conditioned medium
can be obtained also by removing serum from a collected conditioned
medium, using, for example, solvent substitution by dialysis or
column.
[0178] The CNS disease treatment composition is utilized for
treatment of diseases of central nerves (brain and spinal cord).
Examples of CNS diseases to which the CNS disease treatment
composition can be applied include spinal cord injury,
neurodegenerative diseases such as amyotrophic lateral sclerosis,
Alzheimer's disease, Parkinson's disease, progressive supranuclear
palsy, Huntington's disease, multiple system atrophy and
spinocerebellar ataxia, degeneration or loss of neuronal cells
caused by cerebral ischemia, intracerebral hemorrhage or cerebral
infarction, and a retinal disease involving a neuronal cell
disorder. Application of the CNS disease treatment composition
promotes regeneration and healing of CNS tissues due to its neurite
outgrowth effects and/or its apoptosis inhibitory effects toward
neuronal cells. Any disease or disorder to which treatment based on
this mechanism is effective can be the target disease of the
invention, regardless of the type or cause (for example, primary
cause such as external injury or cerebral infarction or secondary
cause such as infection or tumor) of the disease or disorder.
[0179] Spinal cord injury refers to a state in which the spinal
cord is damaged by an external impact or by an internal factor such
as a spinal tumor or hernia, and is classified according to
complete-type (a state in which the spinal cord is completely
severed at a certain point) and incomplete-type (a state in which
the function of the spinal cord is partially maintained although
the spinal cord is damaged or compressed), based on the degree of
the damage. With the current medical technology, complete recovery
from spinal cord injury cannot be achieved, and a new treatment
method is desired to be established. Spinal cord injury is one of
the diseases to which regenerative medicine is expected to be
applied, and use of bone marrow, neural stem cells, embryonic stem
cells, artificial pluripotent stem cells, etc. is under
investigation. However, a decisive treatment technique has not been
realized owing to various problems. Under such a circumstance, the
CNS disease treatment composition provides a treatment method that
is expected to provide a high therapeutic effect, and the
significance and value thereof is quite high.
[0180] Other diseases or disorders to which the CNS disease
treatment composition can be applied include cerebral infarction
caused by degeneration or loss of neuronal cells caused by cerebral
ischemia, intracerebral hemorrhage or the like in the acute phase
or subacute phase, and periventricular leukomalacia, which is a
neonatal encephalopathy caused by hypoxic ischemia during perinatal
period. Cerebral ischemia is a state in which blood supply to the
brain is insufficient, and oxygen and nutrients are not
sufficiently supplied to the brain. Cerebral ischemia causes the
death of neuronal cells and cerebral edema, and serves as a cause
of cerebral infarction. The composition of the invention can be
applied also to the treatment of destruction of neuronal cells due
to cerebral ischemia or the like, or various diseases that
accompany the destruction of neuronal cells.
[0181] Parkinson's disease, spinocerebellar ataxia, Alzheimer's
disease, Huntington's disease, multiple system atrophy and
progressive supranuclear palsy are intractable neural diseases
caused by region-specific neuronal loss in the cerebrum, midbrain
and cerebellum regions. The CNS disease treatment composition is
able to exert a therapeutic effect by suppressing the neuronal
degeneration and loss of in these diseases.
[0182] The CNS disease treatment composition can also be applied to
retinal diseases accompanied by neuronal cell disorders. According
to rough classification, five types of neuronal
cells--photoreceptor cells (cone photoreceptor cells, rod
photoreceptor cells), bipolar cells, horizontal cells, amacrine
cells and ganglion cells--are present in retina. The CNS disease
treatment composition exerts a therapeutic effect by suppressing
the neuronal death and loss in retinal diseases caused by damage to
one type, or two or more types, selected from these neuronal cells
present in retina, as well as in retinal diseases with pathological
conditions exhibiting damage to one type, or two or more types,
selected from these neuronal cells, example of which include
traumatic retinal detachment, retinal tear, concussion of retina,
optic canal fracture, diabetic retinopathy, age-related macular
degeneration, retinitis pigmentosa, glaucoma, choroideremia,
Leber's hereditary optic neuropathy, cone dystrophy, familial
drusen, central areolar choroidal dystrophy and autosomal dominant
optic atrophy.
[0183] Other ingredients may additionally be used in the
composition of the invention, as long as the expected therapeutic
effect is maintained. Ingredients that can additionally be used in
the invention include those listed below.
[0184] (i) Bioabsorbable Materials
[0185] Hyaluronic acid, collagen, fibrinogen (for example, BOLHEAL
(registered trademark)), etc., may be used as organic bioabsorbable
materials.
[0186] (ii) Gelling Materials
[0187] Gelling materials for use preferably have high bioaffinity,
and hyaluronic acid, collagen or fibrin adhesive or the like may be
used. Various hyaluronic acids and collagens may be selected and
used, and it is preferable to adopt those suitable for the purpose
of application of the composition of the invention (the tissue to
which the composition is to be applied). Collagens to be used are
preferably soluble (acid-soluble collagens, alkali-soluble
collagens, enzyme-solubilized collagens, etc.).
[0188] (iii) Others
[0189] Other pharmaceutically-acceptable ingredients (for example,
carriers, excipients, disintegrants, buffer agents, emulsifying
agents, suspending agents, soothing agents, stabilizers,
preservatives, antiseptic agents, physiological saline, etc.) may
be contained. Lactose, starch, sorbitol, D-mannitol, white sugar,
etc. may be used as excipients. Starch, carboxymethylcellulose,
calcium carbonate, etc. may be used as disintegrants. Phosphoric
acid salts, citric acid salts, acetic acid salts, etc. may be used
as buffering agents. Gum arabic, sodium alginate, Tragacanth, etc.
may be used as emulsifying agents. Glycerin monostearate, aluminum
monostearate, methylcellulose, carboxymethylcellulose,
hydroxymethylcellulose, sodium lauryl sulfate, etc. may be used as
suspending agents. Benzyl alcohol, chlorobutanol, sorbitol, etc.
may be used as soothing agents. Propyleneglycol, ascorbic acid,
etc. may be used as stabilizers. Phenol, benzalkonium chloride,
benzylalcohol, chlorobutanol, methylparaben, etc. may be used as
preservatives. Benzalkonium chloride, parahydroxybenzoic acid,
chlorobutanol, etc. may be used as antiseptic agents. Antibiotics,
pH adjusting agents, growth factors (such as nerve growth factor
(NGF), brain-derived neurotrophic factor (BDNF)), etc. may also be
contained.
[0190] The final form of the CNS disease treatment composition is
not particularly limited. Examples of the form include liquid forms
(such as a purely liquid form and a gel form), and solid forms
(such as a powdery form, a fine grain form and a granular
form).
[0191] Methods for producing the CNS disease treatment composition
are not particularly limited. A production method that includes the
following steps (1) to (3) is preferable:
[0192] (1) a step of selecting adhesive cells from dental pulp
cells;
[0193] (2) a step of culturing the adhesive cells; and
[0194] (3) a step of collecting a conditioned medium.
[0195] Each step is described in the following.
[0196] In step (1), dental pulp stem cells, which are adhesive
cells, are selected from dental pulp cells. The dental pulp cells
may be prepared by isolating from a living organism in advance.
This selection step may include preparing dental pulp cells. A
specific example of a procedure of a series of operations from the
preparation of dental pulp cells to the selection of dental pulp
stem cells is described below.
[0197] (a) Collection of Dental Pulp
[0198] A naturally-exfoliated deciduous tooth (or extracted
deciduous tooth or permanent tooth) is disinfected using a
chlorhexidine or ISODINE solution, and, thereafter, the tooth crown
part is divided, and a dental pulp tissue is collected using a
dental reamer.
[0199] (b) Treatment with Enzyme
[0200] The collected dental pulp tissue is suspended in a basal
medium (Dulbecco's Modified Eagle's Medium containing 10% bovine
serum and an antibiotic), and treated with 2 mg/mL collagenase and
DISPASE at 37.degree. C. for 1 hour. The dental pulp cells after
the enzymatic treatment are collected by centrifugation (5,000 rpm)
for 5 minutes. Cell separation using a cell strainer should
basically not be carried out since it decreases the collection
efficiency of neural stem cell fraction such as SHED or DPSC.
[0201] (c) Selection of Adhesive Cells
[0202] The cells are re-suspended in 4 cc of the basal medium, and
seeded in a culture dish for adhesive cells having a diameter of 6
cm. After adding a medium (for example, Dulbecco's Modified Eagle's
Medium (DMEM) containing 10% FCS), the cells are cultured in an
incubator maintained at 5% CO.sub.2 and 37.degree. C. for about two
weeks. After removing the medium, the cells are washed with, for
example, PBS for once or a few times. This operation (the removal
of the medium and the washing of the cells) may be replaced by
harvesting adhesive cells (dental pulp stem cells) that have formed
colonies. In this case, for example, treatment with 0.05%
trypsin-EDTA is carried out at 37.degree. C. for 5 minutes, and
cells that have detached from the dish are harvested.
[0203] In step (2) following step (1), the selected adhesive cells
are cultured. For example, the cells are seeded in a culture dish
for adhesive cells, and cultured in an incubator maintained at 5%
CO.sub.2 and 37.degree. C. Subculture is carried out, if necessary.
For example, when visual observation confirmed that cells has
reached subconfluence (the state in which cells cover about 70% of
the surface area of the culture vessel) or confluence, the cells
are detached from the culture vessel and harvested, and seeded
again into a culture vessel filled with a culture medium.
Subculture may be carried out repeatedly. For example, subculture
may be carried out for one to eight passages, thereby allowing the
cells to proliferate to the required cell number (for example,
about 1.times.10.sup.7 cells/mL). Here, the detachment of cells
from the culture vessel can be carried out using an ordinary method
such as treatment with trypsin. After the culture described above,
the cells may be harvested and stored (in which the storage
condition may be, for example, -198.degree. C.). Cells collected
from various donors may be stored in the form of a dental pulp stem
cell bank.
[0204] The medium may be, for example, a basal medium or a basal
medium supplemented with serum or the like. However, in the case of
preparing a serum-free dental pulp stem cell-conditioned medium, a
serum-free medium may be used throughout the entire process or at
subculturing for the last passage or for the last few passages.
DMEM, Iscove's Modified Dulbecco's Medium (IMDM) (GIBCO
Corporation, etc.), Ham's F12 medium (HamF12) (Sigma-Aldrich
Corporation, GIBCO Corporation, etc.), RPMI1640 medium, etc., can
be used as the basal medium. Two or more basal media may be used in
combination. An example of a mixed medium is a medium formed by
mixing equivalent amounts of IMDM and HamF 12 (commercially
available as, for example, IMDM/HamF12 (tradename, GIBCO
Corporation)). Examples of ingredients that can be added to the
medium include serums (such as fetal bovine serum, human serum and
sheep serum), serum replacements (knockout serum replacement (KSR),
etc.), bovine serum albumin (BSA), antibiotics, various vitamins
and various minerals.
[0205] In step (3) following step (2), the conditioned medium from
the dental pulp stem cells selected and cultured by the
above-described method is collected. For example, the conditioned
medium can be collected by suctioning the culture medium using a
dropper of a pipette. The collected conditioned medium is used as
an active ingredient of the composition of the invention, directly
or after being subjected to one or more treatments. Examples of the
treatments include centrifugation, concentration, solvent
substitution, dialysis, freezing, drying, freeze-drying, dilution,
desalting and storage (for example, 4.degree. C. or -80.degree.
C.). The dental pulp stem cell-conditioned medium exhibited the
expected activity (neurite outgrowth activity and apoptosis
inhibitory activity) even without complex high purification, as
shown in the later-described Examples. This means that the
composition of the invention effective for CNS diseases can be
produced through simple steps. The absence of the necessity for
complex purification step is advantageous also in that a decrease
in activity caused by purification can be avoided.
[0206] In order to confirm the quality of the conditioned medium,
the collected conditioned medium may be subjected to the following
step (a) or step (b), or both.
[0207] (a) a step of checking the conditioned medium with respect
to the presence or absence of a neurite outgrowth activity in the
presence of a nerve regeneration inhibitory substance.
[0208] (b) a step of checking the conditioned medium with respect
to the presence or absence of an apoptosis inhibitory activity
toward neuronal cells.
[0209] A conditioned medium that exhibited a positive result in
step (a) is expected to provide an excellent therapeutic effect by
its neurite outgrowth activity. Similarly, a conditioned medium
that exhibited a positive result in step (b) is expected to provide
an excellent therapeutic effect by its apoptosis inhibitory
activity toward neuronal cells. It is preferable to carry out both
of step (a) and step (b), and use a conditioned medium that
exhibited a positive result in both steps as an active ingredient
of the composition of the invention. Methods for checking in steps
(a) and (b) are as described above (in the section discussing the
first aspect of the invention). The quality of a collected,
prepared or stored conditioned medium can also be checked through
steps (a) and (b). Therefore, it is understood that these steps
themselves have high utility and value as a method of determining
the quality of a dental pulp stem cell-conditioned medium (i.e., as
a means for determining the suitability as an active ingredient for
CNS disease treatment).
[0210] <Method of Concentrating Stem Cell-Conditioned
Medium>
[0211] With regard to the damaged part treatment composition and
the CNS disease treatment composition encompassed by the invention,
physiologically active substances contained in the stem
cell-conditioned medium can be formulated as a drug. This allows,
for example, a nerve regenerative active substance to be formulated
as a drug. For the method of concentrating the cell-conditioned
medium for drug formulation, methods usually employed for this
purpose may be applied. Examples of the concentration method
include the following two methods.
[0212] 1. Spin Column Concentration Method
[0213] The conditioned medium is concentrated (up to 75-fold) using
an AMICON ULTRA CENTRIFUGAL FILTER UNITS-10K (manufactured by
Millipore Corporation). Specific operation procedure thereof is as
described below.
[0214] (i) Add the conditioned medium (up to 15 mL) into an AMICON
ULTRA CENTRIFUGAL FILTER UNITS-10K, and centrifuge at 4000.times.g
for about 60 minutes to concentrate to 200 .mu.l.
[0215] (ii) Add the same amount sterile PBS as the conditioned
medium into the tube, and centrifuge again at 4000.times.g for
about 60 minutes to substitute the basal solution with the PBS.
[0216] (iii) Collect the 200 .mu.l of obtained solution into a
microtube. The collected solution serves as a concentrated stem
cell-conditioned medium.
[0217] 2. Ethanol Precipitation Concentration Method
[0218] The conditioned medium is concentrated (up to 10-fold) using
an ethanol precipitation method. Specific protocol thereof is as
follows.
[0219] (i) Add 45 mL of 100% ethanol to 5 mL of the conditioned
medium, mix the solution well, and left at -20.degree. C. for 60
minutes.
[0220] (ii) Centrifuge at 15,000.times.g at 4.degree. C. for 15
minutes.
[0221] (iii) Remove a supernatant, add 10 mL of 90% ethanol, and
stir well.
[0222] (iv) Centrifuge at 15,000.times.g at 4.degree. C. for 5
minutes.
[0223] (v) Remove a supernatant, dissolve the resultant pellet in
500 .mu.l of sterile water and collect the resultant solution in a
microtube. The collected solution serves as a concentrated stem
cell-conditioned medium.
[0224] <Method of Freeze-Drying Stem Cell-Conditioned
Medium>
[0225] The stem cell-conditioned medium in the composition of the
invention may be freeze-dried. This provides excellent storage
stability. The method of freeze-drying the stem cell-conditioned
medium may be any method usually employed for this purpose.
Examples of the freeze-drying method include the following
method:
[0226] (i) freezing the stem cell-conditioned medium or
concentrated stem cell-conditioned medium obtained by the
above-described method at -80.degree. C. for 2 hours to half a
day.
[0227] (ii) opening the cap of the sample tube after the freezing,
and set the sample tube to a freeze-dryer.
[0228] (iii) freeze-drying the sample for one to two days.
[0229] (iv) obtaining the resultant sample, which serves as a
freeze-dried stem cell-conditioned medium (capable of being stored
at -80.degree. C.).
[0230] A further aspect of the invention provides a method of
treating a CNS disease which includes a step of administering a
therapeutically effective amount of the CNS disease treatment
composition to a CNS disease patient. The administration route of
the composition of the invention is not particularly limited as
long as the composition is delivered to the target tissue. The
composition may be applied, for example, by topical administration.
Examples of the topical administration include injection into the
target tissue or application to the target tissue. The composition
of the invention may be administered by intravenous administration,
intraarterial administration, intraportal administration,
intradermal administration, subcutaneous administration,
intramuscular administration or intraperitoneal administration. The
dosage regimen may be, for example, from once to several times a
day, once every two days, once every three days, or the like. The
dosage regimen may be prepared in consideration of the sex, age,
weight, pathological condition, etc. of the subject (recipient).
The subject to which the composition of the invention is
administered is typically a human patient suffering from a CNS
disease. However, application to mammals other than human
(including pet animals, farm animals and laboratory animals,
specific examples of which include mice, rats, guinea pigs,
hamsters, monkeys, cattle, pigs, goats, sheep, dogs, cats, etc.) is
also contemplated. The composition of the invention is administered
preferably to a subject in the acute or subacute phase, so that the
effects of the composition of the invention are most exerted.
[0231] Simultaneously with or after the administration of the CNS
disease treatment composition, dental pulp stem cells may be
administered to the same subject, thereby providing a complex or
continuous effect, according to an embodiment of the invention.
Here, undifferentiated dental pulp stem cells that have not been
subjected to differentiation inducing treatment after obtainment
thereof, or differentiation-induced dental pulp stem cells that
have been induced to differentiate into a neural cell after
obtainment thereof, may be used as the dental pulp stem cells. In
the case of administering dental pulp stem cells simultaneously
with the administration of the CNS disease treatment composition,
it is preferable to administer undifferentiated dental pulp stem
cells in order that high therapeutic effects are exerted. In the
case of administering dental pulp stem cells after the
administration of the CNS disease treatment composition, it is
preferable to use differentiation-induced dental pulp stem cells
that have been induced to differentiate into neural cells. It is
also possible to use pluripotent stem cells (such as iPS cells or
ES cells) that have been induced to differentiate into neural cells
in addition to, or in place of, the differentiation-induced dental
pulp stem cells.
[0232] Examples of the invention are described below, but not
limited thereto. In the examples, "%" is based on weight (mass)
unless otherwise specified.
EXAMPLES
Example 1
Materials and Methods
[0233] (1) Subjects and Cell Cultures
[0234] Human dental pulp tissues were obtained from clinically
healthy extracted deciduous teeth and permanent teeth from eight
patients. These experimental protocols were approved by the ethics
committee of Nagoya University. SHED and DPSCs were isolated and
cultured as described in Proc Natl Acad Sci USA 2000; 97: 13625-30
or Proc Natl Acad Sci USA 2003; 100: 5807-12.
[0235] Briefly, the pulp was gently removed and digested in a
solution of 3 mg/mL collagenase type I and 4 mg/mL dispase at
37.degree. C. for 1 hour. After filtration using 70-mm cell
strainers (Falcon; BD Labware, Franklin Lakes, N.J.), cells were
cultured in Dulbecco's Modified Eagle Medium (DMEM; GIBCO,
Rockville, Md.) containing 20% mesenchymal cell growth supplement
(Lonza Inc., Walkersville, Md.) and antibiotics (100 U/mL
penicillin, 100 mg/mL streptomycin and 0.25 mg/mL amphotericin B;
GIBCO) at 37.degree. C. with 5% CO.sub.2. After primary culture,
the cells were subcultured at about 1.times.10.sup.4
cells/cm.sup.2. Cells passaged from once to three times were used
in the experiments. Human BMMSCs were purchased from Lonza Inc.,
and cultured according to the manufacturer's instructions.
[0236] (2) Analysis of Cell Proliferation
[0237] The proliferation rates of SHED, DPSCs and BMSCs were
assessed by bromodeoxyuridine (BrdU) incorporation for 12 hours
using a BrdU staining kit (Invitrogen, Carlsbad, Calif.) according
to the manufacturer's instructions (n=3 for each group). The
experiment was repeated five times. Statistically significant
differences were evaluated by the Tukey-Kramer test following
one-way analysis of variance.
[0238] For STRO-1 immunofluorescence, SHED, DPSCs and BMSCs were
fixed with 3% paraformaldehyde, and then rinsed twice with
phosphate-buffered saline, and treated with 100 mM glycine for 20
minutes. Cells were then permeabilized with 0.2% Triton-X
(Sigma-Aldrich, St. Louis, Mo.) for 30 minutes, and subsequently
incubated in a mixture of 5% donkey serum and 0.5% bovine serum
albumin for 20 minutes. Next, the cells were incubated with a mouse
anti-human STRO-1 antibody (1:100; R&D, Minneapolis, Minn.) as
a primary antibody for 1 hour, incubated for 30 minutes with a goat
anti-mouse immunoglobulin M-FITC antibody (1:500; Southern Biotech,
Birmingham, Ala.) as a secondary antibody, and mounted using
Vectashield with DAPI (Vector Laboratories Inc, Burlingame,
Calif.).
[0239] (3) Animal Experiment (FIG. 1)
[0240] Five-week-old female hairless mice (Hos: HR-1) were provided
from SLC Inc. (Shizuoka, Japan). All mice were housed in
climate-controlled quarters (22.+-.1.degree. C. at 50% humidity)
with a 12/12-hour light/dark cycle. Animals were allowed free
access to water and a chow diet, and were observed daily. The mice
were irradiated dorsally using a UVB-emitting system RMX-3W (Handok
Biotech, Seoul, Korea) for eight weeks, five times a week. A bank
of 10 Toshiba SE lamps was used without any filtering for UVB (peak
of emission being about 312 nm, and the irradiance between 290 and
320 nm corresponding to 55% of the total amount of UVB). The
distance from the lamps to the animals' backs was 89 cm. During
exposure, the animals were allowed to move around freely in their
cages. The irradiation dose was 1 MED (minimal erythemal dose; 60
mJ/cm.sup.2) in the first two weeks, 2 MEDs (120 mJ/cm.sup.2) in
the third week, 3 MEDs (180 mJ/cm.sup.2) in the forth week, and 4
MEDs (240 mJ/cm.sup.2) in the fifth through eight weeks. The total
UVB dose was approximately 115 MEDs (6.9 J/cm.sup.2). Five weeks
after wrinkle induction, SH-CM (100%) was subcutaneously injected
into the restricted area of the mice. In a positive control,
PBS-suspended SHED (4.times.10.sup.5) was injected directly into
the dermis. In a negative control, the dermis was treated by PBS
only.
[0241] (4) Preparation of SH-CM
[0242] SHED (4.times.10.sup.5 cells) were cultured in DMEM/F12
(Invitrogen-Gibco-BRL, Grand Island, N.Y.) serum-free medium.
Conditioned medium of SHED was collected after 72 hours of culture,
centrifuged at 300.times.g for 5 min, and filtered using a 0.22 mm
syringe filter.
[0243] (5) Skin Replica and Image Analysis
[0244] At the time of wrinkle induction and one week after the
injection, negative replicas of the dorsal skin surface were taken
using a silicon-based impression material, FLEXTIME1 (Heraeus
Kulzer, New York, N.Y.). To obtain replicas of the wrinkles from
the same skin area, the skin was marked using an oil-based marker
pen. Five weeks after the final injection of SH-CM or SHED to the
skin, impressions were taken from the marked area. For ease of
measurement, all replicas were cut into square pieces of 1 cm, and
the back of each replica was processed into a flat plane using the
same impression material. Light was directed at an angle of
208.degree., and images were incorporated from the replica using a
CCD camera. The image of the negative replicas was observed using a
wrinkle analysis system, skin visiometer SV 600 (Courage &
Khazaka, Cologne, Germany). The parameters used in the assessment
of the skin wrinkles are number, depth and area thereof.
[0245] (6) Histology
[0246] Dorsal skins (1 cm.times.1 cm) were fixed with a 10%
formalin neutral buffered solution, embedded in polyester wax, and
sectioned at 6 mm. The sections were subjected to Hematoxylin &
Eosin (H&E) staining and Masson's trichrome staining.
[0247] (7) HDF Culture and UVB Irradiation Dose
[0248] HDFs were cultured in a DMEM supplemented with 10% fetal
bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin at
37.degree. C. with 5% CO.sub.2. After starvation with serum-free
medium for 24 hours, cells were washed with PBS, and exposed to UVB
with 3 to 4 drops of PBS. UVB irradiation was carried out using a
UV light source (Waldmann, Schwenningen, Germany). Immediately
after the irradiation, the PBS was aspirated, and replaced with a
complete medium. UVB irradiation doses were varied in the range of
from 50 to 250 mJ/cm.sup.2 during the test, and finally fixed to 70
mJ/cm.sup.2 for further experimentation.
[0249] (8) Cell Proliferation Assay
[0250] HDFs were plated at a density of 5.times.10.sup.3 cells/well
in 96-well plates, and the proliferation of HDFs was measured using
a CCK-8 Kit (Dojindo, Gaithersburg, Md.). After starvation for 24
hours in a serum-free medium, the cells were continuously cultured
for 24 hours with or without SH-CM, and were exposed to UVB (70
mJ/cm.sup.2) for 90 seconds. Then, UVB-irradiated cells were
cultured in a complete medium for 24 hours and harvested. HDFs were
added to 10 mL of the CCK-8 solution, and incubated for 3 hours.
The absorbance was measured at 450 nm using a microplate reader
(TECAN, Gro dig, Austria). OD values of each well were converted
into their relative cell numbers based on a comparative standard
curve.
[0251] (9) Western Blot Analysis
[0252] HDFs (2.times.10.sup.4 cells/well) were seeded in 24-well
plates, and pretreated as described above. Then, the cells were
lysed in a RIPA buffer (50 mM Tris-HCl, 0.15 M NaCl, 1 mM EDTA, 1%
Triton X-100, 1% SDS, 50 mM NaF, 1 mM Na.sub.3VO.sub.4, 5 mM
dithiothreitol, 1 mg/mL leupeptin and 20 mg/mL PMSF, pH 7.4). Fifty
micrograms of proteins were separated on an 8% SDS-polyacrylamide
gel by electrophoresis. The proteins were transferred to a PVDF
membrane. The membrane was incubated with an anti-collagen type I
antibody (Santa Cruz, Saint Louis, Mo.) and an anti-matrix
metalloproteinase-1 (MMP-1) antibody (Calbiochem, Darmstadt,
Germany). Then, the membrane was washed, and incubated with
horseradish peroxidase-conjugated anti-goat IgG antibody (1:10,000,
Santa Cruz, Saint Louis, Mo.). The blots were reacted with an
immunoglobulin western reagent, and were exposed to X-ray film.
[0253] Results
[0254] (1) Characterization of SHED, DPSCs and BMSCs.
[0255] SHED and DPSCs displayed a fibroblastic morphology
resembling BMSCs (FIG. 2A-C). Immunofluorescence analysis indicated
that SHED, DPSCs and BMSCs contained STRO-1 positive cells (FIG.
2D-F). The proliferation rate of SHED was significantly higher than
those of DPSCs and BMSCs (FIG. 2G).
[0256] (2) SH-CM Alleviated UV-Induced Wrinkles
[0257] During the period of UV exposure, the mice were observed for
fine wrinkling of the skin. However, the SH-CM treated group and
the SHED injected group appeared to have fewer wrinkles than the
PBS group during the treatment (n=8 for each group). In a replica
analysis, FIG. 3 and FIG. 4 show that repeated SH-CM treatment
alleviated the fine wrinkles induced by UVB irradiation. The SHED
injected group showed the same tendency as that of the SH-CH group.
When the inventors measured the parameters for the wrinkles of
replicas with the skin visiometer SV 600, injection of
natural-level (100%) of SH-CM significantly reduced all parameters
for wrinkles. However, SHED-treated skin exhibits higher
effectiveness than the SH-CM group.
[0258] (3) Histological Observation
[0259] The effect of SH-CM on dermal thickness in UVB irradiated
hairless mice, which showed great changes in skin appendages, was
investigated. FIG. 5 shows the histological measurements of the
dermal thickness of the hairless mouse skin by H&E staining.
Collagen fibers are stained as shown in FIG. 5, and the degree of
staining is remarkably high in the SH-CM treated group (A) and the
SHED injected group (B). Measurement of the dermal thickness showed
significant increases in the SHED injected group and the SH-CM
treated group (FIG. 6). Further, a marked increase of collagen
bundles was observed in both groups, but was not observed in the
control group (FIG. 5).
[0260] (4) SH-CM Increased Proliferation of HDFs
[0261] In order to further study the paracrine mechanism with
respect to the alleviation of skin wrinkles by SHED, a cell
proliferation assay was performed in HDFs that had been primarily
cultured with SH-CM. Although UVB irradiation significantly
decreased the proliferation of HDFs, pretreatment with SH-CM showed
a protective effect on HDFs (FIG. 7). SH-CM contains diverse growth
factors, and in a case in which a unique characteristic of the
growth factors is their ability to initiate mitosis of quiescent
cells, it is possible that enhanced proliferation by SH-CM in this
experiment is mediated by the growth factors secreted from
SHED.
[0262] (5) Expression of Collagen Type I and MMP-1
[0263] Since the collagen content in the dermis was significantly
increased in the SH-CM treated hairless mice, protein expressions
of collagen type I and MMP-1 were examined in HDFs after the SH-CM
treatment (FIG. 8). UVB irradiation clearly reduced the protein
expression of collagen type I and induced that of MMP-1. However,
the protein expression of collagen type I was significantly
increased after the SH-CM pretreatment, while that of MMP-1 was
decreased after the SH-CM pretreatment. These results indicate that
the increased collagen content in the dermis of the SH-CM treated
hairless mice was mediated by the stimulation of collagen synthesis
and the inhibition of collagen degradation in the dermal
fibroblasts.
[0264] Discussion
[0265] The characteristics of SHED were compared with those of
DPSCs and BMSCs, which are considered as standard stem cell sources
in tissue engineering and regenerative medicine. The results
indicated that SHED possesses high proliferation ability, which is
enhanced in the presence of an extracellular matrix, suggesting
that SHED is a useful source for stem cell-based therapy. STRO-1
positive cells were found in SHED, DPSCs and BMMSCs. STRO-1 is
known to recognize a trypsin-resistant cell-surface antigen present
on a subpopulation of bone marrow cells, including a predominant
proportion of skeletal stem cell having high growth and
differentiation potential, and colony forming unit fibroblastic
populations. High proliferative capacity is one of the most
critical characteristics of postnatal somatic stem cells. The
proliferation study using BrdU revealed that SHED shows the highest
population rate among SHED, DPSCs and BMSCs. It was previously
reported that micro array analysis revealed that SHED expresses
multiple growth factors such as FGF, TGF-b, CTGF, NGF and BMP
associated with this pathway, at high levels (S. Nakamura, Y.
Yamada et al., Stem Cell Proliferation Pathways Comparison between
Human Exfoliated Deciduous Teeth and Dental Pulp Stem Cells by Gene
Expression Profile from Promising Dental Pulp, JOE, Vol. 35, (11),
1536-1542, 2009). FGF2 was reported as a cytokine that acts to
promote the proliferation of numerous kinds of cells and control
extracellular matrix generation during tissue regeneration and
wound healing.
[0266] Paracrine factors, such as VEGF, KGF or FGF, may be used for
skin regeneration, and this suggests that stem cell transplantation
is also a "cell-based" cytokine therapy. Importantly, conditioned
media containing growth factors can be used in order to avoid the
negative effect of UVB on HDFs. The concept of paracrine effects
mediating at least part of the effects of stem cell therapy is not
inconsistent with previous data. Cell-based cytokine therapy can
provide benefits in wound healing. Keratinocyte differentiation by
SHED-derived growth factors may contribute to re-epithelialization
in wound closure. Further, SHED-derived growth factors can provide
benefits in wound healing, tissue remodeling and skin graft
genesis.
[0267] Photoaging is a complex process having pathologic
similarities to skin wounds. MSCs play a key role in this process,
and interact with keratinocytes, fat cells and mast cells. MSCs are
also a source of extracellular matrix proteins, of which fibrillar
type I and type III collagens are significantly reduced in the
papillary dermis, and their reduction has been shown to correlate
well with the clinical severity of photoaging. This reduction
results from a combination of reduced procollagen biosynthesis and
increased enzymatic breakdown via the actions of MMP. Fisher et al.
showed that UV irradiation induced the synthesis of MMP in human
skin in vivo (Phipps R P, Borrello M A, Blieden T M., Fibroblast
heterogeneity in the periodontium and other tissues, J Periodontal
Res. 1997 January; 32(1 Pt 2):159-165; Fisher G J, Datta S C,
Talwar H S, Wang Z Q, Varani J, Kang S, et al., Molecular basis of
sun-induced premature skin ageing and retinoid antagonism. Nature
1996; 379: 335-339). Among the MMP family, MMP-1, MMP-13 and
membrane-type MMP-14 display collagenolytic activity, and MMP-2 and
MMP-9 were reported to be true elastases. MMP-mediated collagen and
elastin destruction accounts for a large part of the connective
tissue damage that occurs in photodamaged skin (Tsukahara K,
Nakagawa H, Moriwaki S, Takema Y, Fujimura T, Imokawa G.,
Inhibition of ultraviolet-B-induced wrinkle formation by an
elastase-inhibiting herbal extract: implication for the mechanism
underlying elastase-associated wrinkles, Int J Dermatol 2006; 45:
460-468).
[0268] In this study, it was found that SH-CM not only inhibits a
UVB-induced decrease of the type I collagen but also attenuates
UVB-induced MMP-1 expression in HDFs. Wound healing and skin
rejuvenation from photodamage are complex but orderly processes and
are orchestrated via cytokines and growth factors. Therefore, these
data, when combined with the present study, imply that local
cytokine release may be an important factor mediating the
beneficial SHED rejuvenation effects observed after delivery of
SH-CM. Local delivery of SHED may contribute to healing also
through returning circulating stem progenitor cells to the region
of injury.
[0269] In conclusion, under a circumstance in which the application
of SHED for dermal wound healing was still speculative, the
interaction between SHED-derived growth factors and HDFs was
investigated for the first time. SHED exerts effects on HDFs by
causing an increase in collagen synthesis and by activating the
proliferation and migration activity of HDFs, suggesting that SHED
or SH-CM can be used for the treatment of photoaging and wound
healing. The results also suggest that SHED is more suitable for
dermal wound healing compared with MSCs, in terms of properties
thereof. Mainly with secreted growth factors or ECM proteins, SHED
contributes to enhance wound healing potential of HDFs.
Example 2
(1) Preparation of Growth Factor Mixture (Powder)
[0270] Immortalized human mesenchymal stem cells (MSCs: Ronza Co.,
Ltd, USA) were used to prepare a growth factor (GF) admixture. The
cells were cultured for 2 to 8 passages using 10% FSC-containing
DMEM. At a stage of 80% confluence of cells in the culture dish,
the supernatant (culture medium: CM) was sampled. The sampled CM
was then added to ethanol (CM: Ethanol=1:9), and incubated at
-20.degree. C. for 60 min. The CM was concentrated by spinning
(4.degree. C., 1500 rpm, 15 min). The remaining CM was washed with
90% ethanol at -20.degree. C., and then spun again.
[0271] The concentrated CM was freeze-dried, thereby obtaining a
growth factor powder.
(2) Growth Factors in the Powder
[0272] Each growth factor in the powder was analyzed by the
Western-blotting method. The detected growth factors are as
follows: PDGF, VEGF, IGF, KGF, HGF and TGF.
Example 3
Experimental Study of Bone Regeneration by Growth Factor
[0273] (1) Methods
[0274] Four cylinder-shaped bone defects, each having a diameter of
10 mm and a depth of 10 mm, were formed in the dog mandible.
[0275] A titanium implant (3.75 mm in diameter) was inserted into
the center of each defect.
[0276] The spaces around the implant were filled with graft
materials, such as (1) PRP, (2) 100% GF, (3) MSCs
(1.times.1,000,000) and (4) empty defect (control) (FIG. 10).
[0277] (2) Results
[0278] Eight weeks later, the dog was euthanized and the mandible
with the implants was dissected. A histological specimen was made
and BIC (Bone-Implant Contact) was calculated using a light
microscope and an image analyzing system (FIG. 11).
BIC=total length of bone contact to implant surface/total length of
implant surface.times.100(%)
[0279] There was no significant difference in BIC between MSC
(65.0) and GF (58.6), and the respective BIC values in MSC and GF
were much higher than those of the control (26.4) and PRP (44.2)
(FIGS. 12 and 13).
[0280] (3) Conclusion
[0281] In conclusion, growth factors derived from a mesenchymal
stem cell have similar abilities to a living stem cell with respect
to bone regeneration.
[0282] Clinical Case: 56 Years-Old Male
[0283] The sinus lift procedure with installation of two implants
was performed at a post molar region in maxilla. A 100% cell-based
GF with b-TCP (.beta.-tricalcium phosphate) granules was grafted
into the sinus cavity. Eight weeks later, the grafted portion was
successfully filled with new bone, and osteointegration between the
implants and the bone was confirmed by X-ray observation (FIGS. 14
and 15).
Example 4
Experimental Study of Periodontal Tissue Regeneration by Growth
Factor
[0284] (1) Methods
[0285] A two-wall type periodontal defect was made in the distal
portion of molar teeth in the dog mandible (FIG. 16). The defect in
each dog was treated by the following method or procedure (FIGS. 17
and 18).
[0286] 1) Flap operation (FO, Control)
[0287] 2) GTR method
[0288] 3) MSCs (1.times.100,000)
[0289] 4) GF (100%)
[0290] (2) Results
[0291] At 8 weeks after surgery, the dog mandible with molar teeth
and gingiva was dissected, and a histological specimen thereof was
made. By the histological observation, the depth of the pocket
(N.sub.1-JE; length of the epithelium down growth) and the length
of new cementum (N.sub.2-NC) were evaluated using a light
microscope (FIG. 19). These parameters were useful for the
evaluation of the amelioration of periodontal disease. The length
of new cementum was much longer in GF and MSC than in GTR and the
control. Further, the GF and MSC groups showed remarkable
improvement in the depth of pocket, compared with the GTR and
control groups (FIGS. 20 to 22).
[0292] (3) Conclusion
[0293] The MSC-derived growth factor has similar capacity to MSC
themselves in terms of periodontal tissue regeneration.
[0294] Case report: 64 Year-Old Female
[0295] The medial portion of the lower right canine had a deep
periodontal defect having a depth of 7 mm. The aterocollagen sponge
with 100% GF was filled into the defect (FIGS. 23 and 24). Sixteen
weeks later, the defect seemed to be clinically repaired with a
newly formed periodontal tissue (FIG. 25).
[0296] As described above, cytokine therapies have an advantage
over stem cell therapies in terms of safety, stability, easy
manipulation, easy preservation, easy transportation and low
cost.
Example 5
Confirmation of Therapeutic Effects on Cerebral Infarction
[0297] Cerebral Ischemia Model
[0298] All animal experiments were approved by the Institutional
Animal Care and Use Committee (Nagoya University Graduate School of
Medicine). Adult male Sprague Dawley rats (Japan SLC Inc.,
Shizuoka, Japan) weighing 300-400 g were used. The animals were
initially anesthetized with 5% isoflurane (Abbott Laboratories,
North Chicago), and maintained under anesthesia with 1.5%
isoflurane in a mixture of 70% N.sub.2O and 30% O.sub.2. Their
rectal temperature was maintained at 37.degree. C..+-.0.5.degree.
C. on a heating pad. Focal cerebral ischemia was induced by
permanent focal cerebral ischemia (pMCHO) (day 0) (FIG. 26). A 4-0
monofilament nylon suture (Shirakawa, Tokyo, Japan) with the tip
rounded by flame heating and silicone (KE-200, Shin-Etsu Chemical,
Tokyo, Japan) was advanced from the external carotid artery into
the internal carotid artery until it blocked the origin of the MCA.
The regional cerebral blood flow of the MCA territory was measured
using a laser-Doppler flowmeter (Omega FLO-N1: Omega Wave Inc,
Tokyo, Japan) after occlusion. The response was considered positive
and included only if the reduction in regional cerebral blood flow
was greater than 70%.
[0299] Intranasal Administration of SH-CM
[0300] Seventy-two hours after pMCAO (day 3), the rats were again
anesthetized with 5% isoflurane (Abbott Laboratories, North
Chicago), and maintained under anesthesia with 1.5% isoflurane in a
mixture of 70% N.sub.2O and 30% O.sub.2. Their rectal temperature
was maintained at 37.degree. C..+-.0.5.degree. C. on a heating pad.
The animals were randomly divided into three groups, given
SHED-derived conditioned medium (SH-CM) intranasally (n=1, day 16
sacrificed=1) (group I) or phosphate-buffered saline (PBS)
intranasally (n=1, day 16 sacrificed=1) (group II) or pMCAO
operation only (n=5, day 16 sacrificed=5) (group III). SH-CM, which
had been prepared in the same manner as in Example 1, was used in
this experiment. The rats were laid on their backs, their neck were
elevated by rolled-up 4 cm.times.4 cm gauze, and a total of 100
.mu.l per rat was administered in the olfactory pathway using a
Hamilton microsyringe, 10 .mu.l at a time, alternating the
nostrils, with an interval of 2 min between each administration.
During these procedures, the mouth and the opposite nostril were
shut. Intranasal administration was performed everyday during a
period from day 3 to day 15.
[0301] Evaluation of Motor Disability
[0302] A blind test on the rats was carried out on days 1, 3, 6, 9,
12 and 15 using a standardized motor disability scale with slight
modifications. The rat was given 1 point for each of the following
parameters: flexion of the forelimb contralateral to the stroke
when instantly hung by the tail; extension of the contralateral
hindlimb when pulled from the table; and rotation to the paretic
side against resistance. In addition, 1 point was given for
circling motion to the paretic side when trying to walk, 1 point
was given for failure to walk out of a circle of 50 cm in diameter
within 10 seconds, 2 points were given for failure to leave the
circle, within 20 seconds, and 3 points were scored for inability
to exit the circle within 60 seconds. In addition, 1 point each was
given for inability of the rat to extend the paretic forepaw when
pushed against the table from above, laterally, or sideways. The
evaluation according to the motor disability scale was performed 3
times per animal time-point.
[0303] Assessment of Infarct Volume
[0304] The cryosections obtained from samples on day 16 were
stained with Hematoxylin and Eosin. Image J (National Institutes of
Health, ML) was used to determine each infarct area in 20 coronal
sections (20 mm-thick) at 1.00-mm intervals. The entire infarct
area was covered by these 12 coronal sections. Regional infarct
volumes were calculated by summing the infarct areas and
multiplying these areas by the distance between sections (1.00 mm),
followed by remediation for brain edema.
[0305] Results
[0306] Evaluation of Motor Function
[0307] All groups (group I, group II and group III) displayed high
scores for motor function at the early-stage (the scores on day 1
were 8, 9 and 8.2.+-.0.45, respectively, and the scores on day 3
were 8, 9 and 8.6.+-.0.89, respectively). 6 days later, progressive
alleviation in motor disability in the group I on day 6 became
significant as compared with groups II and III (6, 9 and
8.2.+-.0.84, respectively), and more significant on day 9 as
compared with the groups II and III (5, 8 and 8.8.+-.1.0,
respectively) (FIG. 27). Persistent improvement in the group I was
noted on day 12 (4) and day 15 (3), while persistent impairment due
to motor disability (scores above 8) was observed in the groups II
and III on day 15 (9 and 8.25.+-.0.96, respectively).
[0308] Reduction of Infarct Volume
[0309] There was a significant decrease in the infarct volume on
day 16 in the group I (day 16, 54.3 mm.sup.3, n=1), as compared to
the groups II and III (day 16, 192.7 mm.sup.3, n=1; day 16, 222.7
mm.sup.3, n=1) (FIG. 28). These results suggest that the intranasal
administration of SH-CM promoted regeneration.
[0310] As described above, it was found that cytokine therapy has
an excellent restorative effect toward cerebral infarct areas, and
is useful for treatment of cerebral infarction. Similar results
were also confirmed in other rats.
[0311] It was also found that selection of intranasal
administration for cytokine therapy provides less invasiveness, and
exerts a direct effect on ischemic regions after passing the
blood-brain barrier. Further, since the deciduous teeth stem
cell-conditioned medium is considered to contain various
nutritional factors, more rapid restoration as compared with single
administration of a nutritional factor is expected.
Example 6
1. Preparation of Conditioned Medium from Dental Pulp Stem
Cells
[0312] Conditioned media from dental pulp stem cells (SHED and
DPSCs) were prepared according to the following procedure (see,
FIG. 29), and were used in an experiment for verifying the nerve
regeneration effect.
[0313] (i) Culture dental pulp stem cells in a serum (10%
FBS)-containing medium at 37.degree. C. with 5% CO.sub.2 until the
cells in the culture dish reaches 70% to 80% confluence.
[0314] (ii) When reach 70% to 80% confluence, wash the culture dish
twice with PBS, and replace the medium with a serum-free (0% FBS)
medium.
[0315] (iii) Culture at 37.degree. C. with 5% CO.sub.2 for 48
hours.
[0316] (iv) After the 48 hours culture, collect the serum-free
medium into a centrifugation tube.
[0317] (v) Centrifuge the collected serum-free medium at 1,500 rpm
for 4 to 5 minutes to precipitate impurities such as dead
cells.
[0318] (vi) Transfer the supernatant from the centrifuged
centrifugation tube into another centrifugation tube while paying
attention not to suction impurities.
[0319] (vii) Further centrifuge the collected supernatant at
4.degree. C. and 15,000 rpm for 1 minute to precipitate impurities
again.
[0320] (viii) Transfer the supernatant from the centrifuged
centrifugation tube into another centrifugation tube again while
paying attention not to suction impurities
[0321] (ix) obtain the resultant supernatant, which serves as
dental pulp stem cell-conditioned medium.
2. Neural Cells Used for In Vitro Analysis
[0322] PC12 cells, a cell line derived from an immortalized rat
adrenal pheochromocytoma, were used as neural cells. It is known
that the addition of nerve growth factor (NGF), one of neurotrophic
factors, to PC 12 cells induces the outgrowth of axon-like
processes and differentiation into neuron-like cells. Thus, PC12
cells are used as model cells for various in vitro experiments on
nervous system.
3. Neurite Outgrowth Effect and Apoptosis Inhibitory of Dental Pulp
Stem Cell-Conditioned Medium
[0323] (Neurite Outgrowth Experiment and Apoptosis Induction
Experiment Using Nerve Regeneration Inhibitory Substances)
[0324] The neurite outgrowth effect and apoptosis inhibitory effect
of the dental pulp stem cell-conditioned medium were examined in
the presence or absence of a nerve regeneration inhibitory
substance (neurite outgrowth inhibitory factor). CSPG and MAG were
used as nerve regeneration inhibitory substances. The protocol of
the experiment is as described below.
[0325] (1) Neurite Outgrowth Experiment
[0326] (i) Coat a nerve regeneration inhibitory substance (CSPG or
MAG) on a (poly-L-lysine coated) cell culture well at 37.degree. C.
for 24 hours.
[0327] (ii) Seed PC12 cells in the plates coated with the nerve
regeneration inhibitory substance, and culturing them with the
dental pulp stem cell-conditioned medium for 24 hours. As
comparative groups, a serum-free medium, a fibroblast-conditioned
medium and a bone marrow mesenchymal stem cell-conditioned medium
are used.
[0328] (iii) Evaluate the neurite outgrowth of the PC 12 cells
based on a phase-contrast micrograph thereof.
[0329] (2) Apoptosis Induction Experiment
[0330] P12 cells are seeded in the plates coated with the nerve
regeneration inhibitory substance, and are cultured with the dental
pulp stem cell-conditioned medium for 24 hours. The apoptosis of
the cells is evaluated according to the TUNEL assay. A serum-free
medium, a fibroblast-conditioned medium and a bone marrow
mesenchymal stem cell-conditioned medium are used as comparative
groups.
[0331] In the plates coated with nerve regeneration inhibitory
substance (CSPG, MAG), the dental pulp stem cell-conditioned medium
exhibited a stronger neurite outgrowth effect (FIGS. 30 to 33) and
apoptosis inhibitory effect (FIGS. 34 and 35) as compared to other
groups (comparative groups). The dental pulp stem cell-conditioned
medium exhibited a strong neurite outgrowth effect even without the
addition of NGF, which is essential for PC12 cells to differentiate
into neuron-like cells (i.e., the dental pulp stem cell-conditioned
medium exhibited a strong neurite outgrowth effect by itself).
[0332] That is, as shown in FIG. 30, culturing PC12 neuron-like
cells with the dental pulp stem cell-conditioned medium (for 24
hours) results in outgrowth of neurites (i.e., the dental pulp stem
cell-conditioned medium exhibits neurite outgrowth activity), even
in the dish coated with a nerve regeneration inhibitory substance
CSPG (see FIG. 30). Addition of the bone marrow mesenchymal stem
cell-conditioned medium or the skin-derived fibroblast-conditioned
medium alone, or addition of Y27632 alone, which inhibits ROCK
activation, does not exhibit such outgrowth activity.
[0333] As shown in FIG. 31, the dental pulp stem cell-conditioned
medium increases the proportion of cells exhibiting neurite
outgrowth, and promotes the formation of longer neurites, even
under conditions in which a nerve regeneration inhibitory substance
CSPG is present.
[0334] As shown in FIG. 32, culturing PC12 neuron-like cells with
the dental pulp stem cell-conditioned medium (for 24 hours) results
in outgrowth of neurites (i.e., the dental pulp stem
cell-conditioned medium exhibits neurite outgrowth activity), even
in the dish coated with a nerve regeneration inhibitory substance
MAG. Addition of the bone marrow mesenchymal stem cell-conditioned
medium or the dermal fibroblast-conditioned medium alone, or
addition of Y27632 alone, which inhibits ROCK activation, does not
exhibit such outgrowth activity.
[0335] As shown in FIG. 33, the dental pulp stem cell-conditioned
medium increases the proportion of cells exhibiting neurite
outgrowth, and promotes the formation of longer neurites, even
under conditions in which a nerve regeneration inhibitory substance
MAG is present.
[0336] As shown in FIGS. 34 and 35, almost all of the PC12
neuron-like cells cultured for 24 hours in the dish coated with a
nerve regeneration inhibitory substance MAG or CSPG underwent
apoptosis. The dental pulp stem cell-conditioned medium almost
perfectly inhibits the apoptosis thereof.
[0337] The symbols in FIG. 30 represent the following: PLL:
poly-L-lysine coat, PLL+NGF: poly-L-lysine coat and addition of
nerve growth factor (NGF), PLL/CSPG: poly-L-lysine coat and CSPG
coat, PLL/CSPG Y27632: poly-L-lysine coat, CSPG coat and addition
of Y27632, PLL/CSPG SHED-CM: poly-L-lysine coat, CSPG coat and
culturing with SHED-conditioned medium, PLL/CSPG DPSC-CM:
poly-L-lysine coat, CSPG coat and culturing with DPSC-conditioned
medium, PLL/CSPG BMSC-CM: poly-L-lysine coat, CSPG coat and
culturing with bone marrow mesenchymal stem cell-conditioned
medium, PLL/CSPG Fibro-CM: poly-L-lysine coat, CSPG coat and
culturing with fibroblast-conditioned medium.
[0338] The symbols in FIG. 31 represent the following: PLL:
poly-L-lysine coat, PLL+NGF: poly-L-lysine coat and addition of
nerve growth factor (NGF), PLL/CSPG: poly-L-lysine coat and CSPG
coat, PLL/CSPG+NGF: poly-L-lysine coat, CSPG coat and addition of
NGF, PLL/CSPG+SHED-CM: poly-L-lysine coat, CSPG coat and culturing
with SHED-conditioned medium, PLL/CSPG+NGF+SHED-CM: poly-L-lysine
coat, CSPG coat, addition of NGF and culturing with
SHED-conditioned medium, PLL/CSPG+DPSC-CM: poly-L-lysine coat, CSPG
coat and culturing with DPSC-conditioned medium,
PLL/CSPG+NGF+DPSC-CM: poly-L-lysine coat, CSPG coat, addition of
NGF and culturing with DPSC-conditioned medium, PLL/CSPG+BMSC-CM:
poly-L-lysine coat, CSPG coat and culturing with bone marrow
mesenchymal stem cell-conditioned medium, PLL/CSPG+NGF+BMSC-CM:
poly-L-lysine coat, CSPG coat, addition of NGF and culturing with
bone marrow mesenchymal stem cell-conditioned medium,
PLL/CSPG+Fibro-CM: poly-L-lysine coat, CSPG coat and culturing with
fibroblast-conditioned medium, PLL/CSPG+NGF+Fibro-CM: poly-L-lysine
coat, CSPG coat, addition of NGF and culturing with
fibroblast-conditioned medium, PLL/CSPG+Y27632: poly-L-lysine coat,
CSPG coat and addition of Y27632, PLL/CSPG+NGF+Y27632:
poly-L-lysine coat, CSPG coat, addition of NGF and addition of
Y27632.
[0339] The symbols in FIG. 32 represent the following: PLL:
poly-L-lysine coat, PLL+NGF: poly-L-lysine coat and addition of
nerve growth factor (NGF), PLL/MAG: poly-L-lysine coat and MAG
coat, PLL/MAG Y27632: poly-L-lysine coat, MAG coat and addition of
Y27632, PLL/MAG SHED-CM: poly-L-lysine coat, MAG coat and culturing
with SHED-conditioned medium, PLL/MAG DPSC-CM: poly-L-lysine coat,
MAG coat and culturing with DPSC-conditioned medium, PLL/MAG
BMSC-CM: poly-L-lysine coat, MAG coat and culturing with bone
marrow mesenchymal stem cell-conditioned medium, PLL/MAG Fibro-CM:
poly-L-lysine coat, MAG coat and culturing with
fibroblast-conditioned medium.
[0340] The symbols in FIG. 33 represent the following: PLL:
poly-L-lysine coat, PLL+NGF: poly-L-lysine coat and addition of
nerve growth factor (NGF), PLL/MAG: poly-L-lysine coat and MAG
coat, PLL/MAG+NGF: poly-L-lysine coat, MAG coat and addition of
NGF, PLL/MAG+SHED-CM: poly-L-lysine coat, MAG coat and culturing
with SHED-conditioned medium, PLL/MAG+NGF+SHED-CM: poly-L-lysine
coat, MAG coat, addition of NGF and culturing with SHED-conditioned
medium, PLL/MAG+DPSC-CM: poly-L-lysine coat, MAG coat and culturing
with DPSC-conditioned medium, PLL/MAG+NGF+DPSC-CM: poly-L-lysine
coat, MAG coat, addition of NGF and culturing with DPSC-conditioned
medium, PLL/MAG+BMSC-CM: poly-L-lysine coat, MAG coat and culturing
with bone marrow mesenchymal stem cell-conditioned medium,
PLL/MAG+NGF+BMSC-CM: poly-L-lysine coat, MAG coat, addition of NGF
and culturing with bone marrow mesenchymal stem cell-conditioned
medium, PLL/MAG+Fibro-CM: poly-L-lysine coat, MAG coat and
culturing with fibroblast-conditioned medium, PLL/MAG+NGF+Fibro-CM:
poly-L-lysine coat, MAG coat, addition of NGF and culturing with
fibroblast-conditioned medium, PLUMAG+Y27632: poly-L-lysine coat,
MAG coat and addition of Y27632, PLL/MAG+NGF+Y27632: poly-L-lysine
coat, MAG coat, addition of NGF and addition of Y27632.
[0341] The symbols in FIG. 34 represent the following: PLL:
poly-L-lysine coat, PLL/CSPG: poly-L-lysine coat and CSPG coat,
PLL/CSPG SHED-CM: poly-L-lysine coat, CSPG coat and culturing with
SHED-conditioned medium, PLL/CSPG DPSC-CM: poly-L-lysine coat, CSPG
coat and culturing with DPSC-conditioned medium, PLL/CSPG BMSC-CM:
poly-L-lysine coat, CSPG coat and culturing with bone marrow
mesenchymal stem cell-conditioned medium, PLL/CSPG Fibro-CM:
poly-L-lysine coat, CSPG coat and culturing with
fibroblast-conditioned medium, PLL/CSPG+Y27632: poly-L-lysine coat,
CSPG coat and addition of Y27632, PLL: poly-L-lysine coat, PLL/MAG:
poly-L-lysine coat and MAG coat, PLL/MAG SHED-CM: poly-L-lysine
coat, MAG coat and culturing with SHED-conditioned medium, PLL/MAG
DPSC-CM: poly-L-lysine coat, MAG coat and culturing with
DPSC-conditioned medium, PLL/MAG BMSC-CM: poly-L-lysine coat, MAG
coat and culturing with bone marrow mesenchymal stem
cell-conditioned medium, PLL/MAG Fibro-CM: poly-L-lysine coat, MAG
coat and culturing with fibroblast-conditioned medium, PLL/MAG
Y27632: poly-L-lysine coat, MAG coat and addition of Y27632.
[0342] The symbols in FIG. 35 represent the following: PLL:
poly-L-lysine coat, PLL/CSPG: poly-L-lysine coat and CSPG coat,
PLL/CSPG SHED-CM: poly-L-lysine coat, CSPG coat and culturing with
SHED-conditioned medium, PLL/CSPG DPSC-CM: poly-L-lysine coat, CSPG
coat and culturing with DPSC-conditioned medium, PLL/CSPG BMSC-CM:
poly-L-lysine coat, CSPG coat and culturing with bone marrow
mesenchymal stem cell-conditioned medium, PLL/CSPG Fibro-CM:
poly-L-lysine coat, CSPG coat and culturing with
fibroblast-conditioned medium, PLL/CSPG+Y27632: poly-L-lysine coat,
CSPG coat and addition of Y27632, PLL: poly-L-lysine coat, PLL/MAG:
poly-L-lysine coat and MAG coat, PLL/MAG SHED-CM: poly-L-lysine
coat, MAG coat and culturing with SHED-conditioned medium, PLL/MAG
DPSC-CM: poly-L-lysine coat, MAG coat and culturing with
DPSC-conditioned medium, PLL/MAG BMSC-CM: poly-L-lysine coat, MAG
coat and culturing with bone marrow mesenchymal stem
cell-conditioned medium, PLL/MAG Fibro-CM: poly-L-lysine coat, MAG
coat and culturing with fibroblast-conditioned medium, PLL/MAG
Y27632: poly-L-lysine coat, MAG coat and addition of Y27632.
[0343] As described above, a surprising fact that the dental pulp
stem cell-conditioned medium suppresses the action of nerve
regeneration inhibitory substances, promotes neurite outgrowth, and
suppresses apoptosis, was revealed. In other words, it was revealed
that the dental pulp stem cell-conditioned medium is quite
effective for CNS regeneration and the treatment of CNS
diseases.
[0344] 4. Verification Using Spinal Cord-Injured Model Animal
[0345] (1) Improvement of Motor Function of Hindlimbs by
Administration of Conditioned Medium
[0346] 10th thoracic vertebrae were removed from 8-week-old female
SD rats under general anesthesia with pentobarbital sodium, and
crush injury damage was induced by applying a 200 kilodyn force
from outside the dura mater using an IH impactor, to obtain a model
of spinal cord crush injury. From immediately after the crush
injury, a silicone tube connected to a perfectly implantable
microinfusion pump in which a dental pulp stem cell-conditioned
medium (SHED-CM), a bone marrow mesenchymal stem cell-conditioned
medium (BMSC-CM) or PBS (control) was charged, was inserted from
the subarachnoid cavity below the 12th thoracic vertebra, and
placed so as to allow outflow from directly above the injury site.
The administration was continuously carried out at a flow rate of
24 .mu.l/day for 8 weeks until the rats were sacrificed, and the
motor function of hindlimbs was evaluated every week. The
Basso-Beattie-Bresnahan (BBB) score (Basso D M, Beattie M S,
Bresnahan J C. A sensitive and reliable locomotor rating scale for
open field testing in rats. J. Neurotrauma., 1995:12:1-21.) was
used for the evaluation.
[0347] <BBB Score>
[0348] 0. No observable movement of the hip joint, knee joint and
foot joint at all.
[0349] 1. Slight movement of one or two of the joints.
[0350] 2. Extensive movement of one joint only.
[0351] 3. Extensive movement of two joints only.
[0352] 4. Slight movement of all of the three joints.
[0353] 5. Slight movement of two joints and extensive movement of
the remaining one joint.
[0354] 6. Extensive movement of two joints and slight movement of
the remaining one joint.
[0355] 7. Extensive movement of all of the three joints.
[0356] 8. Sweeping with no weight support, or plantar placement of
the paw with no weight support.
[0357] 9. Very occasional sweeping or stepping with weight support
by the hindlimbs.
[0358] 10. Occasional (5% to 50%) weight supported steps and no
forelimb-hindlimb coordination.
[0359] 11. Frequent (50% to 100%) weight supported steps and no
forelimb-hindlimb coordination.
[0360] 12. Occasional (5% to 50%) weight supported steps and
occasional (5% to 50%) forelimb-hindlimb coordination.
[0361] 13. Frequent (50% to 100%) weight supported steps and
frequent (50% to 100%) forelimb-hindlimb coordination.
[0362] 14. Frequent plantar weight supported steps with
forelimb-hindlimb coordination/consistent plantar weight supported
steps with external rotation of paw position (due to weak muscle
strength).
[0363] 15. Forelimb-hindlimb coordination, occasional (5% to 50%)
steps with heel lifting, and external rotatation of paw
position.
[0364] 16. Forelimb-hindlimb coordination, frequent (50% to 100%)
steps with heel lifting, and occasional parallel positioning of paw
to the body.
[0365] 17. Forelimb-hindlimb coordination, frequent (50% to 100%)
steps with heel lifting, andparallel positioning of paw to the
body.
[0366] 18. Forelimb-hindlimb coordination, steps with heel lifting,
and parallel positioning of paw to the body.
[0367] 19. Forelimb-hindlimb coordination, steps with heel lifting,
parallel positioning of paw to the body, the tail remains down.
[0368] 20. Forelimb-hindlimb coordination, steps with heel lifting,
parallel positioning of paw to the body, and lifting the tail.
[0369] 21. Forelimb-hindlimb coordination, steps with heel lifting,
parallel positioning of paw to the body, lifting of the tail, and
weight support.
[0370] The ameliorating effects of the hindlimb motor function was
compared based on the BBB scores. The evaluation results are shown
in FIG. 36. The group administred with the dental pulp stem
cell-conditioned medium (SHED-CM) exhibited surprising improvement
and recovery of the hindlimb motor function as represented by score
15 (forelimb-hindlimb coordination, occasional (5% to 50%) steps
with heel lifting, and external rotation of paw position). Although
the group administered with the bone marrow mesenchymal stem
cell-conditioned medium (BMSC-CM) also exhibited a certain degree
of improvement, the improvement effect thereof is far lower than
that of the SHED-CM group.
[0371] (2) Alteration of Spinal Cord Morphology After 8 Weeks
[0372] 8 weeks after the initiation of the administration of
SHEM-CM (or BMSC-CM or PBS), the rats were perfusion fixed with
paraformaldehyde. Subsequently, a length of the spinal cord
including the injury site and extending 5 mm rostrally and 5 mm
caudally from the injury site was dissected, and taken out. The
weights of the spinal cords were compared between the Sham group,
the control group, the BMSC-CM group and the SHED-CM group.
[0373] The morphological alteration of the spinal cord 8 weeks
after the initiation of the administration was assessed. The states
of the spinal cords taken out are shown in the upper panel of FIG.
37. The comparison of the weights (masses) of the spinal cords is
shown in the lower panel of FIG. 37. In the SHED-CM treated group,
the atrophy of the spinal cord caudal to the injury site (injury
epicenter) was suppressed (the upper panel of FIG. 37). In other
words, the morphological alteration of the injured spinal cord was
suppressed by the administration of SHED-CM. In accordance with
this result, the SHEM-CM group also exhibited an increase in the
weight of the spinal cord (the lower panel of FIG. 37).
[0374] (3) Spinal Neuroaxis after 8 Weeks
[0375] 8 weeks after the initiation of the administration of
SHEM-CM (or PBS), the rats were perfusion fixed with
paraformaldehyde. A length of the spinal cord including the injury
site and extending 5 mm rostrally and 5 mm caudally from the injury
site was dissected, and taken out. Then, the spinal cord was
embedded and frozen in O.C.T compound, and frozen section slides of
the spinal cord were prepared. The frozen section slides of the
spinal cord were immunostained with an anti-serotonin (5-HT)
antibody and with an antibody against neuroaxis
(anti-Neurofilament-M (NF-M) antibody).
[0376] The damaged portion and a neighbourhood thereof were
histologically examined 8 weeks after the initiation of the
administration. The results of the immunostaining are shown in FIG.
38. Continuous administration of a small amount of SHED-CM
maintained the number of the total neurofilaments (NF-M) in the
region caudal to the injury site. The number of serotonin fibers
projecting from the raphe nuclei of the brain stem to the spinal
cord was also maintained. It was revealed that the loss of
neurofilaments is suppressed by the administration of SHED-CM, and
that a neurotransmitter serotonin produced in the upper areas and
the brain stem was transported to the areas lower than the injury
site.
[0377] (4) Experiment on Apoptosis Suppression by Conditioned
Medium
[0378] The control group and the SHED-CM group were perfusion fixed
with paraformaldehyde 24 hours after the spinal cord crush injury
and one week after the spinal cord crush injury. A length of the
spinal cord including the injury site and extending 5 mm rostrally
and 5 mm caudally from the injury site was dissected, and taken
out. Then, the spinal cord taken out was embedded and frozen in
O.C.T compound, and frozen section slides of the spical cord were
prepared. The frozen section slides of the spical cord were
double-immunostained with TUNEL, which specifically reacts with
fragmentalized DNAs, and an anti-GFAP antibody specific for
astrocytes, an anti-NeuN antibody specific for neuronal cells or an
anti-CNPase antibody specific for oligodendrocytes, to compare the
cell death of neuronal cells and glia cells.
[0379] The cell death of the neuronal cells in the damaged portion
and in the neighbourhood thereof was evaluated 24 hours after the
spinal cord crush injury and one week after the spinal cord crush
injury. The results are shown in FIG. 39. Continuous administration
of a small amount of SHED-CM suppressed apoptotic cell death of
astrocytes, neurons and oligodendrocytes that occurred immediately
after the nerve injury. In spinal cord injury, apoptotic cell death
of oligodendrocytes are observed in a larger area one week after
the injury (enlargement of secondary damage). It was revealed that
SHED-CM also suppresses this apoptotic cell death, thereby
suppressing the enlargement of neural damage.
[0380] From the above, spinal cord injury, neurodegenerative
diseases such as amyotrophic lateral sclerosis, Alzheimer's
disease, Parkinson's disease, progressive supranuclear palsy,
Huntington's disease, multiple system atrophy and spinocerebellar
ataxia, degeneration or loss of neuronal cells caused by cerebral
ischemia, intracerebral hemorrhage or cerebral infarction and a
retinal disease involving a neuronal cell disorder are contemplated
as diseases to which the CNS disease treatment composition
according to the invention can be applied.
[0381] Therefore, according to the invention, since a stem
cell-conditioned medium that is obtained by culturing stem cells
and that contains a mixture of cytokines is used, endogenous stem
cells in the target tissue is allowed to differentiate and
proliferate. As a result, the target tissue is repaired and
regenerated through the proliferation of cells in the damaged part,
the generation of extracellular matrix, etc.
[0382] The invention is by no means limited to the embodiments and
examples of the invention described above. Various modifications
are also included in the invention as long as they are within the
scope of the claims, and can easily be conceived therefrom by those
skilled in the art.
[0383] U.S. Provisional Application No. 61/317,713, filed Mar. 26,
2010, U.S. Provisional Application No. 61/410,370, filed Nov. 5,
2010, Japanese Patent Application No. 2010-267962, filed Dec. 1,
2010, U.S. Provisional Application No. 61/437,697, filed Jan. 31,
2011 and Japanese Patent Application No. 2011-037028, filed Feb.
23, 2011, are incorporated by reference herein in their
entirety.
[0384] All publications, patent applications, and technical
standards mentioned in this specification are herein incorporated
by reference to the same extent as if each individual publication,
patent application, or technical standard was specifically and
individually indicated to be incorporated by reference.
* * * * *