U.S. patent application number 11/377610 was filed with the patent office on 2006-09-21 for internally administered therapeutic agents for cranial nerve diseases comprising mesenchymal cells as an active ingredient.
This patent application is currently assigned to RENOMEDIX INSTITUTE, INC.. Invention is credited to Hirofumi Hamada, Osamu Honmou.
Application Number | 20060210544 11/377610 |
Document ID | / |
Family ID | 37010590 |
Filed Date | 2006-09-21 |
United States Patent
Application |
20060210544 |
Kind Code |
A1 |
Honmou; Osamu ; et
al. |
September 21, 2006 |
Internally administered therapeutic agents for cranial nerve
diseases comprising mesenchymal cells as an active ingredient
Abstract
Intravenous administration of bone marrow cells collected from
rat bone marrow or peripheral blood to a rat cerebral infarction
model was found to be effective in treating cerebral infarction.
Human and murine bone marrow stem cells showed similar effects.
Mesenchymal cells such as bone marrow cells, cord blood cells, or
peripheral blood cells can be used as agents for in vivo
administration against cranial nerve diseases.
Inventors: |
Honmou; Osamu; (Hokkaido,
JP) ; Hamada; Hirofumi; (Hokkaido, JP) |
Correspondence
Address: |
FOLEY AND LARDNER LLP;SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
RENOMEDIX INSTITUTE, INC.
ASKA PHARMACEUTICAL CO., LTD.
MITSUI SUMITOMO INSURANCE CARE NETWORK CO., LTD.
HITACHI LTD.
|
Family ID: |
37010590 |
Appl. No.: |
11/377610 |
Filed: |
March 17, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10562202 |
|
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PCT/JP04/09386 |
Jun 25, 2004 |
|
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11377610 |
Mar 17, 2006 |
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Current U.S.
Class: |
424/93.21 ;
435/368; 435/372 |
Current CPC
Class: |
A61P 7/00 20180101; A61K
2035/124 20130101; A61K 35/28 20130101; G01N 2800/285 20130101;
G01N 2800/2871 20130101 |
Class at
Publication: |
424/093.21 ;
435/372; 435/368 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 5/08 20060101 C12N005/08 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 27, 2003 |
JP |
2003-185260 |
Dec 26, 2003 |
JP |
2003-432329 |
Claims
1. A composition of isolated and purified peripheral blood-derived
mesenchymal stem cells (PMSCs), wherein the PMSCs are over 70%
positive for CD73, essentially 100% positive for CD90, essentially
100% negative for CD45 and essentially 100% negative for CD106.
2. A method preparing peripheral blood-derived mesenchymal stem
cells (PMSCs) comprising: a) obtaining peripheral blood from a
subject or patient, b) optionally diluting the peripheral blood, c)
optionally incubating the peripheral blood, d) centrifuging the
peripheral blood in order to form a supernatant and a cell
fraction, e) discarding the supernatant from the cell fraction, f)
suspending the cell fraction in a culture medium, g) plating the
suspended cells on a surface for tissue culturing, h) incubating
the suspended cells for about 48 hours, wherein the cells form into
a group of cells adhering the surface and a group of cells not
adhering to the surface, i) eliminating the nonadherent cells, j)
further incubating the adherent cells, k) optionally detaching and
subculturing the adherent cells, l) detaching the adherent cells,
m) suspending the adherent cells in a neural progenitor basal
medium, n) plating the cells wherein PMSCs are formed, and o)
harvesting the PMSCs.
3. The method of claim 2, wherein the peripheral blood is diluted
in a red blood cell lysis solution.
4. The method of claim 2, wherein the peripheral blood is
centrifuged at 300-500 G for about 2 to 5 minutes.
5. The method of claim 2, wherein the angle of the centrifuge tube
is 40- 50.degree. and the speed of the centrifuge is between about
1200 to about 3500 rpm.
6. The method of claim 2, wherein the PMSCs are over 70% positive
for CD73, essentially 100% positive for CD90, essentially 100%
negative for CD45 and essentially 100% negative for CD106.
7. A method of preparing peripheral blood-derived mesenchymal stem
cells (PMSCs) comprising: a) obtaining peripheral blood from a
subject or patient, b) optionally diluting the peripheral blood, c)
optionally incubating the peripheral blood, d) centrifuging the
peripheral blood in order to form a supernatant and a cell
fraction, e) discarding the supernatant from the cell fraction, f)
suspending the cell fraction in a culture medium, g) plating the
suspended cells on a surface for tissue culturing, h) incubating
the suspended cells for about 48 hours, wherein the cells form into
a group of cells adhering the surface and a group of cells not
adhering to the surface, i) eliminating the nonadherent cells, j)
further incubating the adherent cells, k) optionally detaching and
subculturing the adherent cells, l) detaching the adherent cells,
m) suspending the adherent cells in a neural progenitor basal
medium, n) plating the cells wherein PMSCs are formed, o)
incubating the cells while optionally adding growth factors,
wherein floating neurospheres of PMSCs are formed, p) collecting
the neurospheres of PMSCs, q) dissociating the of PMSCs cells of
the neurospheres, r) plating the dissociated neurospheres, and s)
culturing the PMSCs.
8. The method of claim 7, wherein after the non-adherent cells are
eliminated, the adherent cells are incubated until reaching or
nearly reaching confluence.
9. The method of claim 7, wherein the neurospheres are centrifuged
at 300-500 G for about 2 to 5 minutes.
10. The method of claim 9, wherein the neurospheres are centrifuged
in a neural progenitor base medium.
11. The method of claim 7, wherein the PMSCs are over 70% positive
for CD73, essentially 100% positive for CD90, essentially 100%
negative for CD45 and essentially 100% negative for CD106.
12. A method of treating a patient who has suffered a stroke or who
suffers from neural lesions caused by injury or disease, ischemia,
infarction, Krabbe's disease, Hurler's syndrome, metachromatic
leukodystrophy, or encephalomyelitis by injecting into the patient
a therapeutically effective amount of the cell fraction of claim
1.
13. The method of claim 12, wherein the injection is
intravenous.
14. The method of claim 13, wherein the site of the injection is
the patient's arm or leg.
15. The method of claim 12, wherein the patient has suffered a
stroke.
16. The method of claim 12, wherein the encephalomyelitis is
autoimmune encephalomyelitis.
17. A method of treating a patient who has suffered a stroke or who
suffers from neural lesions caused by injury or disease, ischemia,
infarction, Krabbe's disease, Hurler's syndrome, metachromatic
leukodystrophy, or encephalomyelitis by injecting into the patient
a therapeutically effective amount of the cell fraction of claim
2.
18. The method of claim 17, wherein the injection is
intravenous.
19. The method of claim 18, wherein the site of the injection is
the patient's arm or leg.
20. The method of claim 17, wherein the patient has suffered a
stroke.
21. The method of claim 17, wherein the encephalomyelitis is
autoimmune encephalomyelitis. 1568834.2
Description
[0001] This application is a continuation-in-part of Application
Ser. No. 10/562,202, which is a national phase application of
PCT/JP04/009386 filed on Jun. 25, 2004. The entire of contents of
Application No. 10/562,202, include the specification, drawing,
claims, sequence listing and abstract are hereby incorporated by
reference.
TECHNICAL FIELD
[0002] The present invention relates to cranial nerve disease
therapeutic agents for in vivo administration, which comprise
mesenchymal cells, particularly bone marrow cells, cord blood
cells, or peripheral blood cells, or cells derived from these cells
as active ingredients.
BACKGROUND ART
[0003] In recent years regenerative medical techniques have been in
the limelight. In regenerative medical techniques, disorders that
the natural, inherent regenerative-healing ability of the human
body cannot cure can be cured by regenerating organs and such using
artificial proliferation of autologous cells, and then surgically
conjugating these at the site of the lesions. Such cures have been
successful in a wide variety of fields.
[0004] Transplantation of oligodendroglia (oligodendrocytes) (see
Non-patent Documents 1 to 3), or myelin-forming cells, such as
Schwann cells (see Non-patent Documents 4, 2, and 5) or olfactory
ensheathing cells (see Non-patent Documents 6 to 8), can elicit
remyelination in animal models and electrophysiological function
may be recovered (see Non-patent Documents 9 and 5). It is not
impossible to prepare such cells from patients or other persons for
use in cell therapy; however, it is problematic since tissue
material must be collected from either the brain or nerves.
[0005] Neural progenitor cells or stem cells derived from the brain
have the ability to self-proliferate, and are known to
differentiate into neurons and glial cells of various lineages (see
Non-patent Documents 10 to 13). Upon transplantation into newborn
mouse brains, human neural stem cells collected from fetal tissues
differentiate into neurons and astrocytes (see Non-patent Documents
14 to 16), and can remyelinate axons (Non-patent Document 17).
There have been reports of the remyelination and recovery of
impulse conduction when neural progenitor cells derived from adult
human brains are transplanted into demyelinated rodent spinal
chords (Non-patent Document 18)
[0006] These studies have evoked great interest since they indicate
the possibility of applying the above-mentioned cells in reparative
strategies for neurological diseases (see Non-patent Documents 18,
14 to 16, and 19).
[0007] Recent studies have revealed that neural stem cells can
produce hematopoietic cells in vivo, indicating that neural
progenitor cells are not limited to nervous system cell lineages
(see Non-patent Document 20). Further, when bone marrow
interstitial cells are injected into newborn mouse lateral
ventricles, they differentiate, to a very small extent, into cells
expressing astrocyte markers (see Non-patent Document 21). Under
appropriate cell culture conditions bone marrow interstitial cells
are reported to produce a very small number of cells that express
nervous system cell markers in vitro; however, it is unclear
whether these cells are useful for neural regeneration (see
Non-patent Document 22).
[0008] The present inventors have previously extracted and cultured
nervous system cells (neural stem cells, neural progenitor cells)
from adult human brains, and established some cell lines. By
studying the functions of these cells, the inventors discovered
that neural stem cells are pluripotent and can self-reproduce (see
Non-patent Document 18). Specifically, single-cell expansion of
neural progenitor (stem) cells obtained from adult human brains was
conducted to establish cell lines; the established cells were then
subjected to in vitro clonal analysis. The results demonstrated
that the cell lines were pluripotent (namely, had the ability to
differentiate into neurons, astroglia (or astrocytes), and
oligodendroglia (i.e., oligodendrocytes)) and had self-reproducing
ability (namely, proliferation potency). Thus, these cells were
confirmed to possess the characteristics of neural stem cells.
[0009] Transplantation of cultured neural stem cells, which were
extracted from small amounts of neural tissue collected from the
cerebrum of an individual, into a lesion of the brain or spinal
cord of the individual, seems to be a widely applicable therapeutic
method in autotransplantation therapy. However, although it doesn't
cause symptoms of neurological deficiency, collecting tissues that
contain neural stem cells from the cerebrum is not easy. Thus,
considering the current need to establish therapeutic methods for
various complicated diseases of the nervous system, it is crucial
to establish safer and simpler methods for autotransplantation
therapy. Thus, to obtain donor cells, the present inventors have
developed techniques for collecting mononuclear cell fractions and
the like from bone marrow cells, cord blood cells, or fetal liver
cells, which is simpler than collecting neural stem cells (see
Patent Document 1). Specifically, the present inventors have shown
that mononuclear cell fractions prepared from bone marrow cells
have the ability to differentiate into nervous system cells. They
also have shown that cell fractions containing mesodermal stem
cells (mesenchymal stem cells), stromal cells, and AC 133-positive
cells, which were separated from the mononuclear cell fraction,
also had the ability to differentiate into nervous system
cells.
[0010] Cranial nerve diseases can be treated by directly
administering an affected part in the brain with cells that have
the ability to differentiate into the above-mentioned nervous
system cells. This technique, however, is very complicated and
dangerous. There is therefore much demand for the development of
simple and safe methods and agents for treating cranial nerve
diseases.
[0011] Mesenchymal stem cells (MSCs) are thought to represent a
very small proportion of cells in the mononuclear population of
bone marrow. These cells will grow to confluency in appropriate
culture conditions as flattened fibroblast-like cells, and have
been suggested to differentiate into bone, cartilage, cardiac
myocytes and neurons and glia both in vitro and in vivo. MSCs
prepared from human bone marrow (BMSCs) have been used in clinical
studies for metachromatic leukodystrophy, Hurler syndrome,
myeloablative therapy for breast cancer [11], graft-versus-host
disease, and stroke.
[0012] Human mesenchymal precursor cells found in the blood of
normal subjects proliferated in culture with an adherent-spread
morphology, and displayed cytoskeletal, cytoplasmic and surface
markers (CD34.sup.-, CD45.sup.-, and CD105.sup.+) of mesenchymal
precursors. These cells had a capacity for differentiation into
fibroblast, osteoblast, and adipocyte lineages. A canine
CD34-fibroblast-like cell in the peripheral blood showed
mesenchymal stem cell characteristics. Because peripheral blood is
readily accessible, stem cells isolated from blood may be a good
candidate for a cell therapy.
[0013] Transplantation of mesenchymal stem cells derived from bone
marrow (BMSCs) after ischemia onset can reduce infarction size and
improve fuictional outcome in rodent cerebral ischemia models.
While intravenous injection of BMSCs reduces infarction size and
improves functional outcome in a rat stroke model, the therapeutic
benefit of MSC-like multipotent precursor cells derived from
peripheral blood (PMSCs) transplantation in cerebral ischemia is
still uncertain.
[0014] Although the potential of MSCs in peripheral blood (PMSCS)
has been studied, it is not previously been known whether
peripheral blood-derived plastic-adherent stem/precursor cells
(PMSCs) can differentiate into a neural lineage or provide a
therapeutic benefit for victims of stroke. [Patent Document]WO
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the domiciliation of mesenchymal stem cells after infusion into
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transplantation of bone marrow stromal cells after cerebral
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D, Schwarz EJ, Prockop DJ, et al. Adult rat and human bone marrow
stromal cells differentiate into neurons. J Neurosci Res 2000;61
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al. Telomerized human multipotent mesenchymal cells can
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cells. Exp Hematol 2003;31:715-722. [Non-patent document 48]
Prockop DJ, Gregory CA, Spees JL. One strategy for cell and gene
therapy: Harnessing the power of adult stem cells to repair
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SUMMARY OF THE INVENTION
[0015] One aspect of this invention is a composition of peripheral
blood-derived mesenchymal stem cells (PMSCs) that is over 70%
positive for CD73, essentially 100% positive for CD90, essentially
100% negative for CD45 and essentially 100% negative for CD106. The
term essentially 100% means nearly all the detectable cells, such
as 99% or more, display or do not display as certain marker.
[0016] Another aspect of this present invention is a method of
differentiating PMSCs into neural cells by [0017] a) obtaining
peripheral blood from a subject or patient, [0018] b) optionally
diluting the peripheral blood, [0019] c) optionally incubating the
peripheral blood, [0020] d) centrifuging the peripheral blood in
order to form a supernatant and a cell fraction, [0021] e)
discarding the supernatant from the cell fraction, [0022] f)
suspending the cell fraction in a culture medium, [0023] g) plating
the suspended cells on a surface for tissue culturing, [0024] h)
incubating the suspended cells for about 48 hours, wherein the
cells form into a group of cells adhering the surface and a group
of cells not adhering to the surface, [0025] i) eliminating the
nonadherent cells, [0026] j) further incubating the adherent cells,
[0027] k) optionally detaching and subculturing the adherent cells,
[0028] l) detaching the adherent cells, [0029] m) suspending the
adherent cells in a neural progenitor basal medium, [0030] n)
plating the cells wherein PMSCs are formed, and [0031] o)
harvesting the PMSCs. PMSCs can be harvested by conventional means,
such as by use of a centrifuge. In one embodiment of the present
invention, the PMSCs form floating neurospheres prior to
harvesting.
[0032] It is preferred to centrifuge at 300-500 G for about 2 to 5
minutes. The angle of the centrifuge tube can be about
40.degree.-50.degree. and the speed of the centrifuge can be about
1,200 to about 3,500 rpm. A suspension for separating or culturing
PMSCs can be any commercially available neural progenitor base
medium. Another aspect of the present invention involves a method
of treating a patient who has suffered a stroke or who suffers from
neural lesions caused by injury or disease, ischemia, infarction,
Krabbe's disease, Hurler's syndrome, metachromatic leukodystrophy,
or encephalomyelitis by injecting into the patient a
therapeutically effective amount of PMSCs. The PMSCs can be
injected, preferably injected intravenously. An intravenous
injection can be made anywhere on the patient, such as in the arm
or leg or other conventional locations. The treatment for stroke
can be after a week of the occurrence of the stroke, less then a
week, less than 36 hours, less than 24 hours, less than 12 hours or
less 6 hours. The surface that the cells are cultured on can be any
type of surface suitable for culturing cells such as a plastic or
glass surface, a surface of a culturing dish or a culture medium
surface.
[0033] Disclosure of the Invention
[0034] The present invention was achieved under these
circumstances, and an objective of the present invention is to
provide safe techniques and agents for treating cranial nerve
diseases. More specifically, an objective is to provide agents for
treating cranial nerve diseases for in vivo administration,
particularly for intravenous administration, comprising mesenchymal
cells, particularly bone marrow cells, cord blood cells, or
peripheral blood cells, or cells derived from these cells as an
active ingredient.
[0035] The present inventors made intensive investigations to
achieve the above objectives. Initially, they investigated the
therapeutic effect on cranial nerve diseases of: collecting bone
marrow cells from mouse bone marrow, separating only the
mononuclear cell fraction therefrom, and using this isolated
fraction as donor cells for intravenous administration to a rat
cerebral infarction model. Consequently, they surprisingly found
that not only local administration, but also intravenous
administration of bone marrow cells exhibits a therapeutic effect
on cranial nerve diseases such as cerebral infarction, spinal cord
injuries, and demyelinating diseases.
[0036] The present inventors made further studies on the
therapeutic effects of bone marrow stem cells (mesenchymal stem
cells) on cranial nerve diseases by intravenously administering
them to the above mentioned model animal in the same manner. They
found that intravenous administration of bone marrow stem cells is
very effective for treating cranial nerve diseases.
[0037] They also found that intravenous administration or local
administration of autologous bone marrow cells or mesenchymal stem
cells is effective for treating cranial nerve diseases. Compared to
allotransplantation and xenotransplantation, autotransplantation is
extremely advantageous in terms of therapeutic effects and further
does not require immunosuppressive drugs.
[0038] As described above, the present inventors discovered the
therapeutic effects of intravenously administering mesenchymal
cells (mesenchymal stem cells), particularly bone marrow cells or
mesenchymal stem cells, on cranial nerve diseases. The present
invention has been achieved based on these findings. As shown below
in the Examples, the present inventors have verified the
therapeutic effects on cranial nerve diseases of intravenously
administering the mesenchymal cells of the present invention by
carrying out various medical or biological experiments and detailed
analysis.
[0039] Specifically, mesenchymal cells (mesenchymal stem cells),
and particularly bone marrow cells themselves, can become
intravenously administered agents for therapies of cranial nerve
diseases.
[0040] The above mentioned therapeutic effects are considered to be
synergistic, including the neuroprotective and neural regenerative
effects of bone marrow cells or mesenchymal stem cells.
Accordingly, bone marrow cells or mesenchymal stem cells are
expected to be intravenously administered cranial nerve protectants
or cranial nerve regenerants.
[0041] The present invention relates to agents for treating cranial
nerve diseases that are administered in vivo, and particularly that
are intravenously administered, where the agents comprise
mesenchymal cells, particularly bone marrow cells, cord blood
cells, or peripheral blood cells, or cells derived from these cells
as an active ingredient. The present invention also relates to
agents for in vivo administration that exhibit neuroprotective or
regenerative actions on cranial nerves, which comprise the above
mentioned mesenchymal cells as an active ingredient, and use of the
agents. More specifically, the present invention provides: [1] A
cranial nerve disease therapeutic agent for in vivo administration,
comprising a mesenchymal cell as an active ingredient. [2] The
agent of [1], wherein the cranial nerve disease is cerebral
infarction. [3] An agent for in vivo administration, exhibiting
neuroprotection and comprising a mesenchymal cell as an active
ingredient. [4] An agent for in vivo administration, exhibiting
cranial nerve regeneration and comprising a mesenchymal cell as an
active ingredient. [5] The agent of any one of [1] to [4], wherein
the in vivo administration is intravenous. [6] The agent of any one
of [1] to [5], wherein the mesenchymal cell is: (a) a mesenchymal
cell introduced with a BDNF gene, PLGF gene, GDNF gene, or IL-2
gene; or (b) an immortalized mesenchymal cell introduced with an
hTERT gene. [7] The agent of any one of [1] to [6], wherein the
mesenchymal cell is a mesenchymal stem cell. [8] The agent of any
one of [1] to [6], wherein the mesenchymal cell is a bone marrow
cell, a cord blood cell, or a peripheral blood cell. [9] A method
for treating a cranial nerve disease comprising the in vivo
administration to a patient of a therapeutically effective amount
of the agent of any one of [1] to [8]. [10] The method of [9],
wherein the bone marrow cell is an autologous cell of the patient.
[11] The method of [9] or [10], wherein the cranial nerve disease
is cerebral infarction. [12] The method of any one of [9] to [ 1
1], wherein the in vivo administration is intravenous
administration. [13] The method of any one of [9] to [12], wherein
the mesenchymal cell is a bone marrow cell, a cord blood cell, or a
peripheral blood cell.
[0042] The present invention provides cranial nerve disease
therapeutic agents for in vivo administration, wherein the agents
comprise mesenchymal cells (for example, bone marrow cells, cord
blood cells, peripheral blood cells, mesenchymal stem cells, or
cells derived from these cells) as an active ingredient.
[0043] Herein the term "in vivo administration" generally means
administration at a site other than the head (brain). The in vivo
administration includes intravenous administration, intramuscular
administration, subcutaneous administration, and intraperitoneal
administration, and of these intravenous administration is most
preferred.
[0044] Herein the term "mesenchymal cells" preferably refers to,
for example, bone marrow cells (mononuclear cell fraction of bone
marrow cells; MCF (mononuclear cell fraction)), cord blood cells,
peripheral blood cells, mesenchymal stem cells (MSCs), or cells
derived from these cells. The mesenchymal cells of the present
invention include, for example, mesenchyme-related cells,
mesoblastic stem cells, and so on. Even if cells referred to as
"mesenchymal cells" in the present invention are classified as
cells other than mesenchymal cells in the future, the cells can
still be suitably used in the present invention.
[0045] The stem cells included in bone marrow are hematopoietic
stem cells and "mesenchymal stem cells (MSCs)". Herein "stem cells"
generally mean undifferentiated cells with self-proliferation
ability and the ability to differentiate into cells which have
specific functions in physiological processes, such as the
proliferation and differentiation of cells constituting living
bodies. Hematopoietic stem cells are stem cells that differentiate
into red blood cells, white blood cells, or thrombocytes.
Mesenchymal stem cells may differentiate via neural stem cells into
nerves, differentiate directly into nerves without going via neural
stem cells, differentiate via stromal cells into nerves (but with
low efficiency), differentiate into viscera, differentiate into the
blood vascular system, or differentiate into bone, cartilage, fat,
or muscle.
[0046] The present invention mainly uses mesenchymal stem cells
(MSCs), but there is also the possibility of using hematopoietic
stem cells and other stem cells (progenitor cells) in the body. The
mesenchymal stem cells can be obtained from bone marrow cells,
collected from the bone marrow. Bone marrow cells from which
mesenchymal stem cells are not separated can also be used for the
treatments, as for the mesenchymal stem cells, although the
efficacy of the former is somewhat less the latter.
[0047] Preparing cells such as mesenchymal stem cells from the
peripheral blood is also thought possible. In fact, the present
inventors have successfully induced cultured cells, derived from
cells contained in the peripheral blood, to differentiate into
cells capable of developing cell markers of neural stem cells and
nervous system cells (neurons and glial cells). G-CSF or SCF is not
always necessary when inducing cells derived from the peripheral
blood to differentiate into nervous system cells. Specially, the
present inventors have found that, when mesoblastic stem cells
(mesenchymal stem cells) prepared from a mononuclear cell fraction
separated from bone marrow fluid or umbilical cord blood, or
embryonic stem cells (ES cells), are cultivated in a basal culture
medium, the mesoblastic stem cells (mesenchymal stem cell) or ES
cells are induced to differentiate into neural stem cells, neurons,
or glial cells. Accordingly, cells with functions equivalent to
mesenchymal stem cells can be prepared by cultivating cells from
peripheral blood, and such cells can be used in the present
invention. The "basal culture media" is not limited, as long as
they are regular culture media used in cell cultivation, and they
are preferably DMEM (Dulbecco's modified essential medium) or NPBM
(Neural progenitor cell basal medium: Clonetics). Other components
of the above mentioned basal culture medium are not particularly
limited, and preferably contains F-1 2, FCS, and/or Neural survival
factors (Clonetics), and so on. The concentration within this
culture medium may be, for example, 50% for F-12 and/or 1% forFCS.
The CO.sub.2 concentrationofthe culture medium is preferably 5%,
but is not limited thereto.
[0048] As used herein, the term "mesodermal stem cell" refers to a
cell constituting tissues embryologically categorized into the
class of mesoderm, including blood cells. A "mesodermal stem cell"
is also a cell that can make copies of itself (divide and
proliferate), with the same potency as that of the original cell,
and with the ability to differentiate into all cell types
constituting mesodermal tissues. The mesodermal stem cell
expresses, for example, the cell markers SH2(+), SH3(+), SH4(+),
CD29(+), CD44(+), CD14(-), CD34(-), and CD45(-), but such cells are
not limited to these markers. Furthermore, so-called
mesenchyme-related stem cells are also included in the mesodermal
stem cells of the present invention.
[0049] The above term "mesenchyme-related cell" refers to
mesenchymal stem cells, mesenchymal cells, precursor cells of
mesenchymal cells and cells derived from mesenchymal cells.
[0050] The term "mesenchymal stem cell" refers to stem cells that
can be obtained from bone marrow, peripheral blood, skin, hair
root, muscle tissue, uterine endometrium, blood, cord blood and
primary cultures of various tissues. Furthermore, cells
finctionally equivalent to mesenchymal stem cells obtainable by
culturing cells in the peripheral blood are also comprised in the
mesenchymal stem cells of the present invention.
[0051] Preferred mesenchymal cells in the present invention are
bone marrow cells and bone marrow stem cells (mesenchymal stem
cell). Cord blood cells, peripheral blood cells, and fetal liver
cells are also preferable examples in the present invention.
[0052] A preferred embodiment of bone marrow cells, cord blood
cells, peripheral blood cells, and fetal liver cells in the present
invention is a cell fraction which is isolated from bone marrow
cells, cord blood cells, peripheral blood, or fetal liver and
comprises cells capable of differentiating into nervous system
cells.
[0053] In another embodiment, the cell fraction is a cell fraction
containing mesoblastic stem cells characterized by SH2 (+), SH3
(+), SH4 (+), CD29 (+), CD44 (+), CD14 (-), CD34(-), and
CD45(-).
[0054] Other examples of the cell fraction are cell fractions
containing interstitial cells characterized by Lin(-), Sca-1(+),
CD10(+), CD11D(+), CD44(+) CD45(+), CD71(+), CD90(+), CD105(+),
CDW123(+), CD127(+), CD164(+), fibronectin (+), ALPH(+), and
collagenase-1 (+), or cell fractions containing cells characterized
by AC133(+).
[0055] Cells contained in the above mentioned cell fractions are
preferably cells capable of differentiating into nervous system
cells.
[0056] The cell fractions in the present invention comprise
mononuclear cell fractions, which were separated from bone marrow
cells, and which contain cells characterized by their ability to
differentiate into nervous system cells. Another embodiment is a
mononuclear cell fraction separated from, for example, cord blood
cells, peripheral blood cells, or fetal liver cells which contain
cells characterized by their ability to differentiate into nervous
system cells. Yet another embodiment is mesenchymal stem cells from
the bone marrow that are released into the peripheral blood, and
which are characterized by their ability to differentiate into
nervous system cells. Active substance or agents, for example, can
be used when mesenchymal stem cells are released into the
peripheral blood, but these substances are not always necessary.
Mesenchymal stem cells collected from the bone marrow and those
derived from the peripheral blood possess common characteristics in
the development of markers of neural stem cells and/or nervous
system cells, but differ from each other in some properties, such
as proliferation rate and rate of differentiation induction. The
mesenchymal stem cells for use in the present invention are not
limited to those collected from the bone marrow but also include
those derived from the peripheral blood. Specifically, the
"mesenchymal stem cells" in the present invention include both of
these cells. In the present invention, the mesenchymal stem cells
derived from the peripheral blood may also be simply referred to as
"mesenchymal cells".
[0057] It is unclear whether the differentiation of cells contained
in the cell fractions of the present invention into neural cells is
caused by the transformation of so-called hematopoietic cells into
neural cells, or, alternatively, by the differentiation of immature
cells capable of differentiating into neural cells that are
comprised in bone marrow cells, cord blood cells, or peripheral
blood cells. However, the majority of the cells differentiating
into neural cells are assumed to be stem or precursor cells,
namely, cells having pluripotency and the ability to
self-propagate. Alternatively, the cells differentiating into
neural cells may be stem or precursor cells which have
differentiated to some extent into endoderm or mesoderm.
[0058] Cells in a cell fraction of the present invention do not
have to be proliferated with any trophic factors (but proliferation
in the presence of trophic factors is possible). Thus, these cells
are simple and practical from the standpoint of the development of
autotransplantation technique for nervous system, and are very
beneficial to the medical industry. In general, bone marrow cells,
cord blood cells, or peripheral blood cells (cell fractions) of the
present invention are derived from vertebrates, preferably from
mammals (for example, mice, rats, rabbits, swine, dogs, monkeys,
humans, etc.), but are not especially limited.
[0059] A cell fraction of the present invention can be prepared,
for example, by subjecting marrow cells or cord blood cells
collected from vertebrate animals to density-gradient
centrifugation at 2,000 rpm in a solution for a sufficient time to
ensure separation, depending on specific gravity, and then
recovering the cell fraction with a certain specific gravity in the
range of 1.07 to 1.1 g/ml. Herein, the phrase "a sufficient time to
ensure separation, depending on specific gravity" refers to a time,
typically about ten to 30 minutes, sufficient for the cells to
shift to positions in the solution for density-gradient
centrifugation that accord with their specific gravity. The
specific gravity of the cell fraction to be recovered is within the
range of 1.07 to 1.08 g/ml (for example, 1.077 g/ml). Solutions
such as Ficoll solution and Percoll solution can be used for the
density-gradient centrifugation, but there is no limit thereto.
Furthermore, cord blood cells collected from vertebrate animals may
be prepared in a similar manner as described above, and can be used
as a cell fraction.
[0060] Specifically, first, bone marrow (5 to 10 .mu.l) collected
from a vertebrate animal is combined with a solution (2 ml L- 15
plus 3 ml Ficoll), and then centrifuged at 2,000 rpm for 15 minutes
to isolate a mononuclear cell fraction (approx. 1 ml). The
mononuclear cell fraction is combined with culture solution (2ml
NPBM) to wash the cells, and then the cells are again centrifuged
at 2,000 rpm for 15 minutes. Then, after removing the supernatant,
the precipitated cells are recovered. In addition to the femur,
sources for obtaining a cell fraction of the present invention
include the sternum and the ilium, which constitutes the pelvis.
Any other bone can serve as a source, as long as it is large
enough. A cell fraction of the present invention can also be
prepared from bone marrow fluid stored in a bone marrow bank, or
from cord blood. When using cord blood cells, the cells can be
obtained from cord blood stored in a bone marrow bank.
[0061] Another embodiment of the cell fractions of the present
invention includes mononuclear cell fractions isolated and purified
from bone marrow cells, cord blood cells, or peripheral blood
cells, which contains mesodermal (mesenchymal) stem cells capable
of differentiating into neural cells. A cell fraction containing
mesodermal stem cells can be obtained, for example, by selecting
cells with a cell surface marker, such as SH2 as described above,
from the above-mentioned cell fraction obtained by centrifuging
bone marrow cells, cord blood cells, or peripheral blood cells.
[0062] Furthermore, a cell fraction containing mesodermal stem
cells (mesenchymal stem cells) capable of differentiating into
neural cells can be prepared by subjecting bone marrow cells or
cord blood cells collected from vertebrate animals to
density-gradient centrifugation at 900 G in a solution for a
sufficient time to ensure separation, depending on specific
gravity, and then recovering the cell fraction with a certain
specific gravity within the range of 1.07 to 1.1 g/ml.
[0063] Herein, the phrase "a sufficient time to ensure separation,
depending on specific gravity" refers to a time, typically about
ten to 30 minutes, sufficient for the cells to shift to positions
in the solution for density-gradient centrifugation that accord
with their specific gravity. The specific gravity of a cell
fraction to be recovered varies depending on the type of animal
(for example, human, rat, or mouse) from which the cells have been
derived. Solutions for density-gradient centrifugation include
Ficoll solution and Percoll solution, but are not limited
thereto.
[0064] Specifically, first, bone marrow (25 ml) or cord blood
collected from a vertebrate animal is combined with an equal volume
of PBS solution, and then centrifuged at 900 G for ten minutes.
Precipitated cells are mixed with PBS and then recovered (cell
density =approx. 4x 107 cells /ml) to remove blood components.
Then, a 5-ml aliquot thereof is combined with Percoll solution
(1.073 g/ml), and centrifuged at 900 G for 30 minutes to extract a
mononuclear cell fraction. The extracted mononuclear cell fraction
is combined with a culture solution (DMEM, 10% FBS, 1%
antibiotic-antimycotic solution) to wash the cells, and is
centrifuged at 2,000 rpm for 15 minutes. Finally, the supernatant
is removed, and the precipitated cells are recovered and cultured
at 37.degree. C. under 5% CO.sup.2 atmosphere.
[0065] Another embodiment of a cell fraction of the present
invention is a fraction of mononuclear cells isolated from bone
marrow cells or cord blood cells, which contains stromal cells
capable of differentiating into neural cells. Examples of stromal
cells include cells characterized by Lin(-), Sca-1(+), CD10(+),
CD11D(+), CD44(+), CD45(+), CD71(+),CD90(+), CD105(+), CDW123(+),
CD127(+), CD164(+), fibronectin (+), ALPH(+), and collagenase-1(+).
A cell fraction containing stromal cells can be prepared, for
example, by selecting cells with a cell surface marker, such as Lin
as described above, from the above-mentioned cell fraction obtained
by centrifuging bone marrow cells or cord blood cells. Furthermore,
such cell fractions can be prepared by subjecting bone marrow cells
or cord blood cells collected from vertebrate animals to
density-gradient centrifugation at 800 G in a solution for a
sufficient time to ensure separation, depending on specific
gravity, and then recovering the cell fraction with a certain
specific gravity within the range of 1.07 to 1.1 g/ml. Herein, "a
sufficient time ensuring separation depending on the specific
gravity" indicates a time, typically about ten to 30 minutes,
sufficient for the cells to shift to positions in the solution for
density-gradient centrifugation that accord with their specific
gravity. The specific gravity of the cell fraction to be recovered
is preferably in the range of 1.07 to 1.08 g/ml (for example, 1.077
g/ml). Solutions for density-gradient centrifugation include Ficoll
solution and Percoll solution, but are not limited thereto.
Specifically, first, bone marrow or cord blood collected from
vertebrate animals is combined with an equal volume of a solution
(PBS, 2% BSA, 0.6% sodium citrate, and 1% penicillin-streptomycin).
A 5-ml aliquot thereof is combined with Ficoll +Paque solution
(1.077 g/ml) and centrifuged at 800 G for 20 minutes to obtain a
mononuclear cell fraction. The mononuclear cell fraction is
combined with a culture solution (Alfa MEM, 12.5% FBS, 12.5% horse
serum, 0.2% i-inositol, 20 mM folic acid, 0.1 mM 2-mercaptoethanol,
2 mM L-glutamine, 1 .mu.M hydrocortisone, 1% antibiotic-antimycotic
solution) to wash the cells, and then centrifuged at 2,000 rpm for
15 minutes. After centrifugation the supernatant is removed. The
precipitated cells are collected and then cultured at 37.degree. C.
under 5% CO.sup.2 atmosphere. Another embodiment of a cell fraction
of the present invention is a mononuclear cell fraction containing
cells characterized by AC133(+) which can differentiate into neural
cells, and which is isolated from bone marrow cells, cord blood
cells, peripheral blood cells, or fetal liver tissues. Such cell
fractions can be obtained, for example, by selecting cells with a
cell surface marker of the above-mentioned AC133(+) from the cell
fraction obtained as described above by centrifuging bone marrow
cells, cord blood cells, or peripheral blood cells. Further, in
other embodiments, the cell fractions can be obtained by subjecting
fetal liver tissues collected from vertebrate animals to
density-gradient centrifugation at 2,000 rpm in a solution for a
sufficient time to ensure separation, depending on specific
gravity, then recovering a cell fraction with a specific gravity in
the range of 1.07 to 1.1 g/ml, and then recovering cells with
AC133(+) characteristics from the cell fraction. Herein, "a
sufficient time ensuring separation depending on specific gravity"
refers to a time, typically about ten to 30 minutes, sufficient for
the cells to shift to positions in the solution for
density-gradient centrifugation that accord with their specific
gravity. The solutions for density-gradient centrifugation include
Ficoll solution and Percoll solution, but are not limited
thereto.
[0066] Specifically, first, liver tissue collected from vertebrate
animals is washed in L- 15 solution, and then enzymatically treated
for 30 minutes at 37.degree. C. in an L-15 solution containing
0.01% DNaseI, 0.25% trypsin, and 0.1% collagenase . Then, the
tissue is dispersed into single cells by pipetting. These single
fetal liver cells are centrifuged by the same procedure as that
described for the preparation of mononuclear cell fractions from
femur in Example 1 (1). The cells thus obtained are washed, and
then AC133(+) cells are collected from the washed cells using an
AC133 antibody. Thus, cells capable of differentiating into neural
cells can be prepared from fetal liver tissues. The antibody-based
recovery ofACl33(+) cells can be achieved using magnetic beads or a
cell sorter (FACS, etc.). Transplanting any of these cell fractions
containing mesodermal stem cells (mesenchymal stem cells),
interstitial cells, or AC133-positive cells into demyelinated
spinal cords can lead to efficient remyelination of demyelinated
regions. In particular, the above-mentioned cell fractions
containing mesodermal stem cells (mesenchymal stem cells) can
favorably engraft and differentiate into nervous system cells or
glial cells when transplanted into a cerebral infarction model.
[0067] The cells capable of differentiating into neural cells,
which are contained in the above-mentioned cell fractions, include
for example, neural stem cells, mesodermal stem cells (mesenchymal
stem cells), interstitial cells, and AC133-positive cells which are
contained in the above-mentioned cell fractions, but are not
limited thereto as long as they can differentiate into neural
cells.
[0068] The active ingredients of the cranial nerve disease
therapeutic agents for in vivo administration of the present
invention comprise not only bone marrow cells, cord blood cells, or
peripheral blood cells, but also the above-mentioned cell
fractions. In the present invention it is possible to administer
mesenchymal cells such as bone marrow cells, cord blood cells, or
peripheral blood cells without any modification. However, to
improve the efficiency of therapy, they may be administered as
agents (compositions) to which various agents have been added, or
as cells to which genes with the function of increasing therapeutic
effect have been introduced. The preparation of agents or
transgenic cells of the present invention may comprise, but is not
limited to: [0069] (1) adding a substance that improves the
proliferation rate of cells included in a cell fraction, or that
enhances the differentiation of cells into nervous system cells, or
introducing a gene having the same effect; [0070] (2) adding a
substance that improves the viability of cells in damaged neural
tissues included in a cell fraction, or introducing a gene having
the same effect (e.g., reduction of radicals); [0071] (3) adding a
substance that inhibits the adverse effects of damaged neural
tissues on the cells in a cell fraction, or introducing a gene
having the same effect; [0072] (4) adding a substance that prolongs
the lifetime of donor cells, or introducing a gene having the same
effect (e.g., the hTERT gene); [0073] (5) adding a substance that
modulates the cell cycle, or introducing a gene having the same
effect; [0074] (6) adding a substance aimed at suppressing
immunoreaction, or introducing a gene having the same effect;
[0075] (7) adding a substance that enhances energy metabolism, or
introducing a gene having the same effect; [0076] (8) adding a
substance that improves the migration ability of donor cells in
host tissues, or introducing a gene having the same effect; [0077]
(9) introducing a substance that improves blood flow (including the
induction of angiogenesis), or a gene having the same effect (e.g.,
VEGF, angiopoietin, or PGF); [0078] (10) adding a substance having
neuroprotection activity, or introducing a gene having the same
effect (e.g., BDNF, GDNF, NT, NGF, FGF, EGF, or PFG); [0079] (11)
adding a substance having an apoptosis inhibitory effect, or
introducing a gene having the same effect; or [0080] (12) adding a
substance having an antitumor effect, or introducing a gene having
the same effect (e.g., IL-2 or IF-.beta.).
[0081] The present inventors verified that mesenchymal stem cells
(MSCs) introduced with BDNF (brain-derived neurotrophic factor)
gene, which is a nerve nutritional factor, have therapeutic effects
on a rat cerebral infarction model, as shown in the Examples below.
In addition, the present inventors confirmed that intravenous
transplantation of MSCs introduced with the BDNF gene has
therapeutic effects on cerebral infarction. Similarly, they have
confirmed that intravenous transplantation of MSCs introduced with
PLGF (placental growth factor) gene show therapeutic effects on
cerebral infarction.
[0082] The present inventors have also found that MSCs introduced
with genes other than the BDNF gene, such as the GDNF (glial cell
line-derived neurotrophic factor), CNTF (ciliary neurotrophic
factor), or NT3 (neurotrophin-3) gene, show therapeutic effects on
cerebral infarction. They have verified that mesenchymal stem cells
introduced with IL-2 gene have therapeutic effects on a rat brain
tumor model. Thus, preferred embodiments of the mesenchymal cells
for use in the present invention are mesenchymal cells introduced
with the BDNF gene, PLGF gene, GDNF gene, or the IL-2 gene.
Specifically, mesenchymal cells with an exogenous BDNF gene, PLGF
gene, GDNF gene, or IL-2 gene in an expressible condition are
preferably used as mesenchymal cells in the present invention.
[0083] Apart from the above genes, mesenchymal stem cells
introduced with a gene such as the CNTF or NT3 gene are also
preferred as specific examples of the mesenchymal cells in the
present invention. Hereinafter, a mesenchymal stem cell (MSC)
introduced with the "XX" gene may be referred to as "MSC-XX".
[0084] Combining the mesenchymal cells of the present invention
with factors (genes) that are responsible for angiogenesis is
expected to show significant therapeutic effects on treatments of
cerebral infarction, since cerebral infarction shows symptoms of
vascular occlusion. The present inventors have found that direct
injection of the angiopoietin gene into cerebral infarctions
exhibits significant angiogenetic effects, as shown in the Examples
below. Specifically, MSCs introduced with a gene involved in
angiogenesis, such as the angiopoietin gene, are expected to have
therapeutic effects, particularly on cerebral infarctions.
[0085] Mesenchymal cells introduced with a desired gene in an
expressible manner can be suitably prepared using techniques known
to those skilled in the art.
[0086] The bone marrow fluids to be used in the present invention
can be collected, for example, by anesthetizing (locally or
systemically) vertebrate animals (including humans), puncturing a
bone with a needle, and then aspirating with a syringe. The bones
include, but are not limited to, thefemur, sternum, and osilium,
which forms the pelvis. Further, a procedure that involves directly
puncturing the umbilical cord with a needle, and aspirating with a
syringe to collect and store the cord blood at birth, has also
become an established technique. Bone marrow cells are collected
from subjects under local anesthesia, in an amount of preferably
several milliliters per collection. Please note that this amount
does not apply to case A below, but does apply to cases B and
C.
[0087] Procedures for bone marrow collection include, but are not
limited to, the following procedures:
[0088] (A) Transplanting living bone marrow cells
[0089] When bone marrow cells are collected from humans, for
example, bone marrow fluid is collected by an anesthetist from
patients (the ilium and the like) under general anesthesia, after
sufficient consideration of the safety of general anesthesia. A
target number of cells for collection is 3x 109 or more mononuclear
leukocytes. It is assumed that the target cell number can be
obtained from about 200 ml to about 400 ml of bone marrow fluid.
The upper limit of the bone marrow amount to be collected is
calculated in consideration of patient strain, and is calculated
using the hemoglobin level (Hb level) immediately prior to bone
marrow collection, and body weight of the patient, in consideration
of patient strain. However, in the case of an elderly patient,
where collection of the necessary amount of bone marrow fluid is
problematic, a maximum amount should be collected based on the
decision of the doctor in attendance at the collection of bone
marrow fluid.
[0090] The upper limit of bone marrow to be collected depending on
Hb level immediately prior to collection: [0091] (1) Collect 12 ml
or less per kg of patient body weight, when Hb level is less than
12.5 g/dl; [0092] (2) Collect 15 ml or less per kg of patient body
weight, when Hb level is less than 13.0 g/dl; [0093] (3) Collect 18
ml or less per kg of patient body weight, when Hb level is less
than 13.5 g/dl; or [0094] (4) Collect 20 ml or less per kg of
patient body weight, when Hb level is 13.5 g/dl or more.
[0095] Bone marrow fluid should not be collected when a patient
suffers from cytopenia in the peripheral blood (a white blood cell
count less than 2,000 per milliliter; neutrophil count less than
1,000 per milliliter; hemoglobin level less than 11.0 g/dl;
platelet count less than 10.times.10.sup.4 per milliliter) or when
the patient suffers from hemorrhagic diathesis.
[0096] The collection of bone marrow from patients who use
anticoagulants or antiplatelet agents should be carefully
considered while performing, prior to bone marrow collection and
general anesthesia, hemostasis-coagulation tests (FDP, fibrinogen,
ATIII) which comprise bleeding time and ACT, and that can be
performed as emergency tests.
[0097] (1) Patients using anti-platelet agents (such as Panaldine,
Bufferin, and Bayaspirin):
[0098] Platelet function does not recover until seven days or more
after discontinuation, and bone marrow collection in an acute stage
may cause hemorrhage. Thus, bone marrow collection should be
carefully performed. When bleeding time exceeds ten minutes, bone
marrow collection should not be performed.
[0099] (2) Patents using anti-coagulants (such as warfarin):
[0100] Bone marrow should be collected after ACT has been
normalized by intravenous injection of vitamin K (K1 or K2).
[0101] Bone marrow cells are intravenously administered by mixing
bone marrow cells (3.times.10.sup.9 cells or more) with an equal
amount of a diluent (antibiotic-free RPMI 1,640) and intravenously
injecting the mixture, for example. The entire quantity is expected
to be about 400 to 2000 cc. Administration is as rapid as possible,
but to inhibit coagulation during the administration period,
heparin is generally co-injected.
[0102] For example, 250 cc of the collected bone marrow cell fluid
is mixed with an equal amount of a diluent and 2500 units of
heparin to make up 500 cc, the mixture is immediately filtrated
through a filter to yield an intravenously injectable preparation,
and intravenous administration of the preparation to a patient is
begun immediately. Collection of bone marrow cell fluid is also
continued during this time. This operation is repeated two to six
times, and a set amount of bone marrow cell fluid is administered.
The amount of heparin to be thus administered is about 5,000 to
15,000 units which is substantially the same as the safe and
effective amount for which evidence in the acute stage of cerebral
infarction is already obtained. However, when considering the
continuation of bone marrow collection during administration, ACT
is determined, and treatment such as neutralization with protamine
is conducted as necessary. The time needed for collection is about
two hours. The total amount of bone marrow fluid (including
diluent) to be administered intravenously is about 2000 cc.
Intravenous administration is completed in about three to four
hours while sufficiently monitoring strain to the right heart,
indicated by central venous pressure and so on. Note that about
2000 ml of bone marrow fluid (including diluent) is intravenously
administered, and about 1000 ml of the bone marrow fluid is
collected. Thus the volume of fluid applied to the patient is 1000
ml in three to four hours, which is not so large considering the
volume load applied during conventional treatment of cerebral
infarction.
[0103] Patients are preferably selected according to the following
requirements, but are not limited thereto: [0104] 1. Patients aged
20 to 70; [0105] 2. Patients within 24 hours of onset; [0106] 3.
Patients for whom diffusion-weighted MRIs show abnormalities in the
supratentorial cerebral cortex, perforating region, or both; [0107]
4. Patients whose NINDS-III category is any of atherothrombotic
cerebral infarction, lacunar infarction, or cardiogenic cerebral
embolism; [0108] 5. Patients whose Modified Rankin Scale for the
present episode is 3 or more; [0109] 6. Patents whose impaired
consciousness rates 0 to 100 on the Japan Coma Scale.
[0110] Patients under the following conditions are preferably
excluded. [0111] 1. Patients with improving symptoms and diagnosed
as substantially asymptomatic or as TIA (transient ischemic attack)
patients; [0112] 2. Patients diagnosed as having a causative lesion
of a disorder other than obliterative cerebrovascular disorders,
such as an intracranial hemorrhage, based typically on CT or MRI;
[0113] 3. Patients with cardiogenic embolus where hemophilic
alterations have already been observed by CT; [0114] 4. Patients in
a coma with a severe consciousness disorder of 200 or more on the
Japan Coma Scale; [0115] 5. Pregnant patients or patients at risk
of pregnancy; [0116] 6. Patents with grave renal diseases, liver
diseases, or digestive organ diseases; [0117] 7. Patents with
malignant tumors; [0118] 8. Patients in whom grave abnormalities,
such as severe ischemic heart disease, are suspected in the
cardiovascular system; [0119] 9. Patients meeting the subject
exclusion criteria for bone marrow fluid collection; [0120] 10.
Patients for whom general anesthesia is judged as risky; [0121] 11.
Patients with cerebellar infarction or brainstem infarction; [0122]
12. Patients who have undergone endovascular surgical treatment in
the acute stage; or [0123] 13. Patients judged by the doctor in
charge of treatment as unsuitable subjects for this treatment. (B)
Culturing, preserving, and administering mesenchymal stem cells
(MSCs) collected from the bone marrow fluid and so on:
[0124] Another embodiment of the intravenous administration of the
present invention is, for example, the intravenous administration
of MSCs collected from the bone marrow fluid and so on, then
cultured, and preserved. Preferred conditions for collection,
culture, and preservation of the bone marrow cells are as
follows:
[0125] Specifically: [0126] (1) Collect about 5 ml of bone marrow
fluid from the ilium under local anesthesia; [0127] (2) Extract
MSCs from the collected bone marrow fluid, cultivate and
proliferate, for example, using the method described in WO
02/00849; [0128] (3) Cryopreserve in a preservation medium; [0129]
(4) Thaw the frozen MSCs as needed and intravenously administer
thawed MSCs intact (2 x 108 cells or more).
[0130] In the present invention, bone marrow fluid can be safely
and easily collected under local anesthesia from almost all
patients, since the amount of the bone marrow to be collected in
the initial stages is about 3 ml to about 5 ml, and the strain on
the body is small.
[0131] Since the collected mesenchymal stem cells, such as bone
marrow stem cells, can be proliferated, they can be proliferated in
advance to an amount required for treatment. The MSCs to be used in
the present invention are preferably primary culture MSCs, and more
preferably, primary culture MSCs of 2.times.10.sup.8 cells or more.
The mesenchymal stem cells proliferated by the above methods, or
the therapeutic agents of the present invention can be preserved
for long periods using a predetermined procedure, such as freezing.
Preservation and thawing methods are as follows:
[0132] The cells are thawed in the following manner. First,
equipment and materials such as a program freezer, a freezing bag
F- 100, liquid nitrogen, and a tube sealer are prepared. Reagents
such as Trypsin/EDTA, DMSO, dextran autoserum, and D-MEM are also
prepared.
[0133] After removing the culture medium, T/E is added, adherent
cultured MSC cells are recovered, an equal amount of a cell washing
fluid (D-MEM containing 2% autoserum) is added, and this is then
centrifuged at 400 g for five minutes. The cell pellets are stirred
with a cell-washing fluid (D-MEM containing 2% autoserum) and
centrifuged at 400 g for five minutes. Next, the cells are stirred
in 41 ml of a cell-preservation medium (D-MEM containing 50%
autoserum). In this procedure, two 0.5 ml portions of the cell
suspension are sampled using a 1 ml syringe, and the cells are
counted. The stirred fluid is subjected to bacteriological and
virological examinations to confirm it is uncontaminated by
bacteria or viruses. Next, 10 ml of a cryoprotective fluid (5 ml of
DMSO (Cryoserv) and 5 ml of 10% dextran 40) is added. The resulting
suspension is packed into freezing bags at 50 ml per bag, and the
specimen number is indicated on each bag. The bags are frozen in a
program freezer, and the frozen bags are transferred to and stored
in a liquid nitrogen tank.
[0134] The cells are thawed and washed as follows: First, equipment
and materials such as a warm water bath, a clean work station, a
centrifugal separator, a separating bag, and a tube sealer are
prepared, and reagents such as 20% human serum albumin (or
autoserum), physiological saline, and 10% dextran 40 are prepared.
A freezing bag comprising the cells is removed from the liquid
nitrogen tank and left to stand in the gaseous phase for five
minutes, and at room temperature for two minutes. The bag is left
to stand in the gaseous phase and at room temperature to prevent
its explosion caused by the vaporization of liquid nitrogen. The
bag is placed in a sterilized plastic bag to prevent leakage of its
contents due to, for example, pinholes in the bag. The plastic bag
is placed in a warm water bath and is thawed. After thawing, the
entire quantity of the cell suspension is recovered in a blood bag
(closure system) or tube (open system). The recovered cell
suspension is added to an equal amount of a washing fluid (25 ml of
20% human serum albumin, 75 ml of physiological saline, and 100 ml
of 10% dextran 40). The mixture is left to stand for five minutes
to reach equilibrium, intracellular DMSO is removed, and the
mixture is then centrifuged at 400 g for five minutes. The cell
pellets are stirred with cell-washing fluid. The resulting cell
suspension is administered in vivo to a patient, and again, two 0.5
ml portions of the cell suspension are sampled using a 1 ml syringe
and subjected to a viability assay and bacteriological
examination.
[0135] In the present invention, the primary culture MSCs, which
were collected, cultivated, and preserved in advance, can be
immediately thawed to an active state as needed, and can be
immediately administered intravenously for treatment. Heparin is
not used herein. The patient to be administered has no specific
limitations. (C) Administering mesenchymal stem cells immortalized
by hTERT:
[0136] The present inventors succeeded in developing a method for
stably inducing the differentiation and proliferation of large cell
numbers (WO 03/038075). Generally, mesodermal stem cells
(mesenchymal stem cells) are useful in the medical field of neural
regeneration; however, the proliferation of such cells under
culture conditions is limited to some extent. However, according to
the studies of the present inventors, the in vitro introduction
into stromal cells or mesenchymal stem cells of a viral vector
containing, as an insert, an immortalization gene such as
telomerase, was revealed to result in the continuation of cell
proliferation, even after cycles of cell division, greatly
extending the life span of the cells, and still retaining the same
morphology as normal cells. The present inventors found that
mesodermal stem cells (mesenchymal stem cells) immortalized by
introducing an immortalization gene can be efficiently induced to
differentiate into neural stem cells and nervous system cells under
appropriate culture conditions.
[0137] Specifically, the inventors succeeded in inducing mesodermal
stem cells (mesenchymal stem cells), which had been immortalized
through the introduction of the immortalization gene hTERT, to
differentiate into fat cells, chondroblasts, and osteoblasts, for
example.
[0138] Furthermore, the inventors induced the efficient
differentiation of mesodermal stem cells (mesenchymal stem cells)
immortalized by the introduction of the hTERT gene, into nervous
system cells containing neural stem cells. The present inventors
further revealed that demyelinated areas in the spinal cord can be
repaired by transplanting these cells themselves (the mesodermal
stem cells (mesenchymal stem cells)); neural stem cells
differentiated from the mesodermal stem cells (mesenchymal stem
cells); nervous system cells differentiated from neural stem cells
which had been differentiated from the mesodermal stem cells
(mesenchymal stem cells); and nervous system cells differentiated
from the mesodermal stem cells (mesenchymal stem cells).
[0139] In addition, neural stem cells and nervous system cells
whose differentiation was induced according to the above-described
methods of the present invention, or by the mesodermal stem cells
(mesenchymal stem cells) themselves having an immortalization gene
introduced therein, are expected to be very useful in achieving
neural regeneration.
[0140] When a cell is immortalized by introducing an oncogene or
such, the character of the cell is also transformed. In contrast,
when a cell is immortalized by introducing an immortalization gene,
as in the present invention, the cell retains its original
character. In addition, in cases where an immortalization gene has
been introduced, the gene can be removed after sufficient
proliferation.
[0141] Mesenchymal cells introduced with an immortalization gene
can also be used for intravenous administration in the present
invention as appropriate.
[0142] Since technique (C) can yield a large quantity of cells, a
larger number of cells can be intravenously administered.
Preferably, 1.times.10.sup.9 or more cells can be administered.
This is a tremendous advantage, since therapeutic effect increases
as the number of administered cells increases.
[0143] The cranial nerve disease therapeutic agents for in vivo
administration of the present invention, comprising mesenchymal
cells as an active ingredient, can be formulated according to
methods known to those skilled in the art. For example, the agents
can be used parenterally in the form of an abacterial solution or
suspension for injection, combined with water or other
pharmaceutically acceptable liquid. The agents can be formulated,
for example, by appropriate combination with pharmacologically
acceptable carriers or vehicles (specifically, sterile water,
physiological saline, vegetable oils, emulsifiers, suspending
agents, surfactants, stabilizers, fillers, vehicles, antiseptic
agents, and binders), into the form of generally acceptable unit
dosages as required in drug manufacturing procedures. The amount of
active ingredient in these pharmaceutical preparations is set so as
to yield an appropriate volume within an indicated range. An
aseptic composition for injection can be formulated using a vehicle
such as distilled water for injection, according to regular
preparation procedures.
[0144] Aqueous solutions for injection include, for example,
physiological saline and isotonic solutions containing other
adjuvants such as glucose, D-sorbitol, D-mannose, D-mannitol, or
sodium chloride. These aqueous solutions may be used in combination
with appropriate solubilizers, such as alcohols, more specifically
ethanol and polyalcohols, such as propylene glycol and polyethylene
glycol; and nonionic surfactants such as Polysorbate 80.TM. and
HCO-50.
[0145] Oily liquids include sesame oil and soy bean oil. These can
be used in combination with a solubilizer such as benzyl benzoate
or benzyl alcohol. Buffers such as phosphate buffer and sodium
acetate buffer; soothing agents such as procaine hydrochloride;
stabilizers such as benzyl alcohol and phenol; and antioxidants may
also be combined. Injections prepared in this way are generally
packaged into appropriate ampules.
[0146] In vivo administration of the agents to patients is
preferably parenteral administration. Specifically, it is a single
dose intravenous administration, but can be a multiple dose
administration. The administration can be conducted over a short
period or continuously over a long period. More specifically, the
administration includes injection-type and dermal
administration-type administrations. Injection-type administration
includes intravenous injection, intraarterial injection, selective
intraarterial injection, intramuscular injection, intraperitoneal
injection, hypodermic injection, intracerebroventricular injection,
intracranial injection, and intraspinal injection, and of these
intravenous injection is preferred.
[0147] Intravenous injection enables transplantation by a regular
blood transfusion procedure, does not require surgery or local
anesthesia of the patient, and reduces the burden on both patient
and doctor. Intravenous injection is preferable in that it also
enables bedside transplantation. Considering future advances in
emergency medicine, administration may also be possible during
ambulance transportation or at the scene of an episode.
[0148] Further, due to their high capacity for migration, cells
comprised in a cell fraction of the present invention can be used
as carriers (vectors) for genes. For example, the cells are
expected to be useful as vectors for the gene therapy of various
neurological diseases, such as cerebral infarction and brain
tumor.
[0149] Cranial nerve diseases of the present invention include
cerebral infarction, cerebral stroke, encephalorrhagy,
subarachnoidal hemorrhage, and brain tumor, of which cerebral
infarction is preferred. The cause can be any of atherothrombotic
cerebral infarction, cardiogenic cerebral embolism, and lacunar
stroke categorized in NINDS-III (Classification of Cerebrovascular
Diseases (the third edition) by NINDS (National Institute of
Neurological Disorders and Stroke)). The cranial nerve diseases
also include neurological diseases associated with head injuries,
such as head injuries and cerebral contusion; ischemic cranial
nerve injuries; traumatic cranial nerve injuries; cranial nerve
degenerative diseases; and metabolic nerve diseases, but are not
limited to these, as long as they are diseases caused by
abnormalities in the cranial nerve.
[0150] In vivo administration, such as intravenous administration
of the mesenchymal cells of the present invention enables
neuroprotection in the brain and cranial nerve regeneration.
Accordingly, the present invention provides agents for in vivo
administration that exhibit neuroprotective effects and comprise
mesenchymal cells as active ingredients. The term "neuroprotection"
herein refers to the effect of saving cranial neurons that would be
damaged or die without treatment.
[0151] In addition, the present invention provides agents for in
vivo administration that exhibit cranial nerve regeneration and
that comprise mesenchymal cells as active ingredients. Herein the
term "cranial nerve regeneration" means the effect of regenerating
cranial nerve cells to recover their function, or therapeutic
effects obtained from this effect.
[0152] Furthermore, the present invention relates to methods of
treating cranial nerve diseases, including the in vivo
administration (preferably intravenous) of a therapeutically
effective amount of an agent of the present invention to a
patient.
[0153] To reduce the risk of transplant rejection, mesenchymal
cells such as bone marrow cells, cord blood cells, or peripheral
blood cells in the agents for use in the above mentioned
therapeutic methods are preferably cells collected from the
patient, or cells derived therefrom (autologous cells derived from
the patient) (autotransplantation treatment), unless a special
operation such as immunosuppression is conducted. This is
preferable there is no need for the concomitant use of
immunosuppressive drugs. Allotransplantation is possible if
immunosuppression is carried out; however, autotransplantation
treatments can be expected to exhibit significantly greater
therapeutic effect.
[0154] When autotransplantation treatment is difficult, cells
derived from another person or from another animal for medical use
can be used. These cells may be cryopreserved.
[0155] The autologous cells can be any undifferentiated cells
collected from a patient, cells prepared by subjecting
undifferentiated mesenchymal stem cells collected from a patient to
gene manipulation, and cells prepared by inducing differentiation
of undifferentiated mesenchymal stem cells collected from a
patient.
[0156] The agents of the present invention (mesenchymal cells such
as bone marrow cells) can be suitably administered to patients by
the above-mentioned methods, for example. Doctors can administer
the agents of the present invention to patients by appropriately
modifying the above-described methods.
[0157] The therapeutic methods of the present invention are not
limited to humans. In general, the methods of the present invention
can also be conducted in the same manner, using mesenchymal cells,
on non-human mammals, such as mice, rats, rabbits, pigs, dogs, and
monkeys.
[0158] The inventors have found that fibroblast-like adherent cells
with phenotypic characteristics resembling those of mesenchymal
stem cells prepared from the bone marrow can be cultured from
peripheral blood. These cells showed proliferation and
differentiation into neural lineages in vitro, confirmed by
immunocytochemistory and RT-PCR.
[0159] Mesenchymal stem cell populations obtained from rat
peripheral blood and bone marrow of the rat metaphysis easily
expanded in vitro and exhibited a fibroblast-like morphology. Flow
cytometry analysis to study the surface protein expression on
undifferentiated BMSCs and PMSCs indicated that the myeloid
progenitor antigen CD45 was not expressed by these cells. On the
other hand, PMSCs expressed CD73 (SH3), which has been used to
characterize mesenchymal stem cells. In addition, nestin expression
by PMSCs and BMSCs and their ability to grow in suspension in
defined culture conditions brought them nearer to a neurosphere
phenotype. When nestin-positive neurospheres were dissociated and
plated onto an adherent surface without growth factors, neuronal
and glial differentiation was observed The present inventors have
shown that rat PMSCs proliferated, highly transformed to
nestin-positive neural stem cells (neurospheres), and
differentiated into neuronal or glial cells in vitro. Thus,
autologous peripheral blood is indicated as an important source of
cells for a cell therapy, since they are easy to isolate and expand
for autotransplantation with little risk of rejection.
[0160] The inventors have also shown that intravenous infusion of
MSCs, derived from either bone marrow or peripheral blood, 6 hours
after permanent MCAO in the rat results in reduction in infarction
volume, improvement in cerebral blood flow, induction of
angiogenesis, MSC accumulation in the ischemic brain, and
improvement in behavioral performance. PMSCs derived from
peripheral blood, expanded in culture and intravenously infused
contributed to the therapeutic benefits in the rat MCAO model with
a large effect.
[0161] A characteristic feature of the BMSCs derived from rat bone
marrow is the marker profile of CD45 (-), CD73 (+), CD90 (+), CD106
(-) cell surface phenotype. PMSCs derived from peripheral blood
expressed a similar pattern of cell surface antigens and cellular
morphology (flattened and spindle-shaped adherent cells) in
culture, suggesting similarity of the two cell populations.
[0162] The mechanisms of therapeutic benefits of MSCs
transplantation for stroke, may result from neuroprotection and
angiogenesis. A number of neurotrophic factors have been reported
to have therapeutic effects on cerebral infarction. These include
BDNF, GDNF, NGF, EGF, and bFGF. Mechanisms proposed for the
neuroprotective effect of these agents include anti-apoptotic
activity, free radical scavenging, anti-inflammatory activity, and
anti-glutamate excitotoxicity.
[0163] An advantage of PMSCs for transplantation studies is that
they can be easily and safely obtained in large numbers from blood,
which is a less invasive proceed than extracting from bone
marrow.
[0164] MSCs also provide several angiogenic growth factors such as
VEGF and bFGF, which may prevent endothelial cells from ischemic
damage or stimulate angiogenesis. These cells produce soluble
mediators that down-regulate immune responses which could also
contribute to neuroprotection. Hemodynamic changes of cerebral
blood flow after MCAO with and without MSCs transplantation were
analyzed by PWI. While both control and MSCs transplantation groups
showed improvement of rCBF in the lesion, recovery of rCBF was
greater in the MSC transplantation groups than control groups.
Moreover, histological examination of capillary vessels in ischemic
lesion indicated that MSCs transplantation group showed greater
angiogenesis. These data suggest that the improvement of cerebral
blood perfusion plays an important role in the mechanism of
therapeutic effects of MSC transplantation.
[0165] The present invention can be used to treat neurological
damage caused by injury and neurological diseases including
Krabbe's disease, Hurler's syndrome, metachromatic leukodystrophy,
and stroke. Improved neurological function in experimental
autoimmune encephalomyelitis (EAE) has been reported following
intravenous infusion of human MSCs and neurosphere-derived
multipotent precursors. Suggested mechanisms include reduction of
inflammatory infiltration, remyelination, and elevation of trophic
factors that may be neuroprotective or stimulate
oligodendrogliosis. The present invention may have the advantage of
exerting multiple therapeutic effects at various sites and times
within the lesion as the cells respond to a particular pathological
microenvironment.
[0166] In the working examples, the centrifuge used was one
supplied by Kubota. Cells were injected intravenously into rats by
injection into the femoral vein of the rat in the leg.
[0167] All prior art documents cited herein are hereby incorporated
by reference into this specification.
BRIEF DESCRIPTION OF THE DRAWINGS
[0168] FIG. 1 is a photograph showing the therapeutic effect of
intravenous administration of MCF cells on a rat cerebral
infarction model (transient middle cerebral artery occlusion
model). The intravenously administered MCF cells accumulated in the
cerebral infarction area.
[0169] FIG. 2 is a graph showing the results of investigating the
therapeutic effect of locally and intravenously administered MCFs,
in which open bars indicate local administration and filled bars
indicate intravenous administration.
[0170] FIG. 3 shows photographs depicting the therapeutic effect of
transplanting autologous MCFs (1.times.10.sup.7 cells) to a rat
cerebral infarction model (transient middle cerebral artery
occlusion model: 45 minutes) three hours after cerebral infarction
(A), six hours after cerebral infarction (B), 12 hours after
cerebral infarction (C), 24 hours after cerebral infarction (D), or
72 hours after cerebral infarction (E), or the untreated group
(F).
[0171] FIG. 4 shows a graph of the results of the above-mentioned
FIG. 3, in which open bars indicate data of the untransplanted
group, and filled bars indicate data of the transplanted group.
*<0.001.
[0172] FIG. 5 shows photographs depicting the result of
intravenously administering autologous MCFs (1.times.10.sup.7
cells) to a rat cerebral infarction model, in which A shows
transplanted MCF cells (blue) accumulating in a cerebral infarction
area. B is a photograph obtained by high power magnification of the
region indicated by the open square in A (HE staining). C is a
visualized image of the same region as in B (in blue) after
treatment with x-gal; many transplanted MCF cells (blue) have
accumulated; LacZ-positive cells (D) are found to be NSE-positive
(E), and F is a merged view of D and E; LacZ-positive cells (G) are
found to be GFAP-positive (H), and I is a merged view of G and
H.
[0173] FIG. 6 shows photographs depicting the very frequent
migration of transplant MCF cells into the brain. The cells were
transplanted three hours after cerebral infarction (A), 12 hours
after cerebral infarction (B), or 72 hours after cerebral
infarction (C). D and G show the region indicated by the open
square in A after staining; E and H show the region indicated by
the open square in B after staining; and F and I show the region
indicated by the open square in C after staining.
[0174] FIG. 7 shows graphs indicating the therapeutic effects of
transplantation. (A) shows the results of investigating higher
brain functions (memory and learning) in a Morris water maze test
and (B) is a graph indicating the results of a treadmill stress
test. Filled triangles indicate the untreated group, and open
squares indicate the treated group. *p<0.05, **p<0.01.
[0175] FIG. 8 shows photographs depicting the MRI test results of
rats with cerebral infarction. The upper row shows data immediately
after cerebral infarction and the lower row shows data one week
after cerebral infarction. Scale bar: 5 mm.
[0176] FIG. 9 shows photographs depicting the therapeutic effects
of using MSCs. The upper row shows data immediately after cerebral
infarction and immediately before treatment, and the lower row
shows data after treatment conducted one week after the cerebral
infarction. Scale bar: 5 mm.
[0177] FIG. 10 shows photographs and a graph indicating the results
when MSCs (1.times.10.sup.4 to 1.times.10.sup.7 cells) were
intravenously administered 12 hours after the cerebral
infarction.
[0178] FIG. 11 shows photographs and a graph of results that
histologically support the results of FIG. 10. Compared to the
untreated group (A), a marked therapeutic effect can be seen in the
treated group (B: transplantation of lx 106 cells). (C) shows
quantified histological results.
[0179] FIG. 12 shows photographs indicating the results of
intravenous administration of MSCs (1.times.10.sup.6 cells) to a
rat cerebral infarction model. The transplanted donor cells
accumulated at areas of cerebral infarction (A, B, C, and D).
Photographs A and C are photofluorograms, and photographs C and D
are photofluorograms merged with regular photographs. The
untransplanted group showed no donor cells (E and F). Some of the
transplanted donor cells differentiated into neurons (G, I, and K)
and glial cells (H, J, and L). LacZ-positive cells (G) were found
to be NSE-positive (I). Photograph K shows photograph G merged with
photograph I. LacZ-positive cells (H) were found to be
GFAP-positive (J). Photograph L shows photograph H merged with
photograph J. Scale bars: 250 .mu.m (A and B), 10 .mu.m (C to F),
and 5 .mu.m (G to L).
[0180] FIG. 13 shows photographs and graphs indicating the
therapeutic effects of intravenous MSC administration, as
investigated by magnetic resonance spectroscopy (MRS).
[0181] FIG. 14 shows graphs indicating the therapeutic effects of
MSC transplantation, as investigated by ethological
examination.
[0182] FIG. 15 shows photographs indicating the results of
investigating the therapeutic effects of MSCs on a rat permanent
middle cerebral artery occlusion model.
[0183] FIG. 16 shows photographs indicating that abnormal signals
are also detected in concordance with a cerebral infarction area in
MRI examination of severe cerebral infarction (rat permanent middle
cerebral artery occlusion model).
[0184] FIG. 17 shows photographs indicating that when cerebral
infarction is not treated the clarity of the above-mentioned
abnormal signal of FIG. 16 in cerebral infarction (HIA in MRI)
increases with time (12 hours, three days, and seven days after
cerebral infarction).
[0185] FIG. 18 shows photographs of MRI images showing the results
of intravenous administration of mesenchymal stem cells (MSCs)
(10.times.10.sup.6 cells) to a rat permanent middle cerebral artery
occlusion model. Results are divided according to time elapsed from
the onset of disorder to the administration of MSCs. The images
show data without treatment, and for treatment three hours, six
hours, 12 hours, 24 hours, and 72 hours after onset, indicated
sequentially from the upper row. Each image was obtained by MRI
examination (T.sub.2WI) one week after the onset of cerebral
infarction. The cerebral infarction is white in these images.
[0186] FIG. 19 shows a graph indicating the results of
intravenously administering mesenchymal cells (1.times.10.sup.6
cells) to severe cerebral infarctions (a rat permanent middle
cerebral artery occlusion model), in which the cerebral infarction
area is quantitatively determined in terms of the infarct
volume.
[0187] FIG. 20 shows photographs indicating the therapeutic effect
over time of intravenous MSC administration in the hyperacute stage
of severe cerebral infarction.
[0188] FIG. 21 shows photographs indicating examples of the
therapeutic effect over time of intravenous MSC administration in
the acute stage of severe cerebral infarction.
[0189] FIG. 22 shows a graph of the viability after the onset of
disorder upon intravenous administration of mesenchymal stem cells
(MSCs) (1.times.10.sup.6 cells) to severe cerebral infarction (rat
permanent middle cerebral artery occlusion model). The results are
divided according to the time elapsed from the onset of disorder
until the administration of mesenchymal stem cells. An "n" denotes
the number of samples.
[0190] FIG. 23 shows a graph indicating clinical symptoms after MSC
transplant therapy for severe cerebral infarction.
[0191] FIG. 24 shows photographs of adherent cultured cells, such
as mesenchymal stem cells obtained from the peripheral blood in an
untreated group or in a group pre-administered with G-CSF or SCF
factor by hypodermic injection.
[0192] The left view of FIG. 25 is a photograph showing that it was
possible to induce the differentiation of adherent cultured cells
into neural stem cells (Neurospheres). The right view of FIG. 25 is
a photograph showing that it was also possible to confirm nestin
expression using RT-PCR.
[0193] The upper half of FIG. 26 shows photographs indicating that
the cells shown in FIG. 25 could be induced to differentiate into
neurons (NF-positive cells) and glial cells (GFAP-positive cells).
The lower half of FIG. 26 shows photographs indicating that the
expression of both NF and GFAP could also be confirmed by
RT-PCR.
[0194] FIG. 27 is a graph showing that culturing MSCs results in
BDNF production. MSCs transfected with AxCAhBDNF-F/RGD (MSC-BDNF)
at MOIs of 100, 300, 1000, and 3000 pu/cell secreted
0.230.+-.0.110, 0.434.+-.0.122, 0.931.+-.0.101, and 1.860.+-.0.41
ng/10.sup.5-cells of BDNF, respectively, 48 hours later.
Untransfected MSCs also produced BDNF (0.0407.+-.0.0059 ng/10.sup.5
cell/48-hr).
[0195] FIG. 28 shows graphs indicating evaluations of cerebral
ischemia-induced neural deficiency. [0196] A: Leg placement
impairment was evaluated according to the following scale: 0:
severe neural deficiency, 16: no neural deficiency. The four
ischemia groups showed no statistical difference in leg-placement
score, one day after MCAO and before intracranial administration of
MSCs. Eight days after MCAO, MSC-BDNF-treated rats had
significantly higher leg-placement scores than control DMEM rats
(P=0.0001) and fibroblast-treated rats (P=0.003). Fifteen days
after MCAO, the scores of MSC-BDNF-treated rats had similarly
increased compared to the scores of the DMEM group (P=0.024).
[0197] B: Prior to MCAO the average treadmill speeds were compared
between the groups. Eight days after MCAO, rats in the MSC-BDNF
group achieved a significantly higher speed than those in the
control DMEM (P=0.001) and the fibroblast-treated (P=0.017) group.
The speed in MSC-BDNT group remained different (significantly
higher than the control DMEM (P=0.002) and the fibroblast group
(P=0.023)) until Day 15.
[0198] FIG. 29A is a graph of T2-weighted images (T2W) of rats
administered with DMEM, fibroblasts, MSCs, or MSC-BDNF taken two,
seven, and 14 days after MCAO. Seven days after MCAO, the
MSC-BDNF-treated rats showed a significant reduction in HLV (%) as
compared to rats treated with DMEM (P=0.002), fibroblasts
(P=0.015), or MSCs (P=0.028). Fourteen days after MCAO, the
MSC-BDNF-treated rats showed a significant reduction in HLV (%)
compared with the DMEM-treated rats (P=0.01 1).
[0199] FIG. 29B shows photographs of representative T2W images of
rats administered with DMEM, MSCs, or MSC-BDNF, taken two and seven
days after MCAO. Compared to the other groups on Day 7, the
MSC-BDNF group showed a reduction in ischemic injury volume.
[0200] FIG. 30 is a graph showing in vivo BDNF production levels.
The MSC-BDNF-transplanted rats showed a significantly increased
BDNF level in the ischemic hemisphere seven days after MCAO
compared to rats treated with DMBM (P=0.0002) or MSCs (P=0.0006).
Compared to the DMEM-treated rats, the MSC-treated rats also showed
a significantly increased BDNF level in the ischemic hemisphere
(P=0.0124).
[0201] FIG. 31 shows diagrams indicating the presence of cells
having a DNA fragment in the ischemic penumbra and at the site of
application after MCAO. [0202] A: Photographs showing that compared
to the DMEM-treated rats, the MSC-BDNF-treated rats had virtually
no TUNEL-positive cells. FITC =green (TUNEL-positive), P=red
(nucleus), magnification .times.200. [0203] B: Photographs A
magnified by 630 times.
[0204] FIG. 31 C is a graph showing that animals treated with
MSC-BDNF in the ischemic boundary zone showed a significant
reduction in TUNEL-positive cells compared to DMEM-administered
animals (P=0.0 13).
[0205] FIG. 31D shows photographs indicating that fewer positive
cells were detected in the MSC-BDNF-treated rats than in the
MSC-treated rats. A large number of DsR-positive MSCs were detected
within 2 mm of the administration site. FITC (green,
TUNEL-positive), DsR (red, MSC).
[0206] FIG. 32 shows micrographs indicating morphological
characteristics of exogenous MSCs and endogenous brain cells in the
rat brain. Double-immunofluorescence staining revealed that EGFP
cells were localized near the administration site. In the brains of
recipient rats, EGFP cells (green), neurogenic nucleus antigen
(NeuN; A) and glial fibrillary acidic protein (GFAP; B) were found
using confocal laser scanning microscopy. Scale bar: 20 .mu.m.
[0207] FIGS. 33a to 33e are graphs showing the expression of
surface antigens in rat MSCs analyzed by flow cytometry. The MSCs
were labeled with monoclonal antibodies specific to the antigen to
be presented. Dead cells were removed by front and side scattering.
FIGS. 33f to 33i are photographs indicating the differentiation of
rat MSCs into typical mesenchymal cells. Osteogenic differentiation
of primary MSCs or MSC-IL2s was detected by von Kossa staining.
Adipogenic differentiation of primary MSCs (h) or MSC-IL2s (i) was
detected by Oil Red 0 staining.
[0208] FIG. 34 shows graphs indicating the antitumor effect and
migration capability of MSCs. Filled bars represent NRK cells, and
open bars represent MSC cells. (a): 9L cells (5.times.10.sup.4
cell/well) were co-cultured with MSC or NRK cells (5.times.10.sup.3
cell/well). (b): MSC or NRK cells were inoculated at a
concentration of 1.times.10.sup.5 cells in a Transwell Insert, and
9L cells (5.times.10.sup.3 cell/well) were placed in wells. The 9L
cells were counted four days later. All data are expressed by
proliferation inhibitory percentage (%) =[1 -(number of 9L cells
co-cultured with MSC or NRK cells/number of 9L cells cultivated
alone)].times.100. Graph (c) shows the results of a migration
assay. .sup.1251-deoxyuridine-labeled cells (5.times.10.sup.4)
isolated using a filter of 8 .mu.m pore size were then placed in
the upper chamber of a Transwell, and 9L cells were placed in the
lower chamber. After 24-hours of incubation, radioactivity in the
lower chamber was determined. The results of the cell migration
assay are expressed as ratios of the cell number in the lower
chamber to the total cell number in the chamber.
[0209] FIG. 35 shows photographs indicating the distribution and
migration of MSCs in rats with glioma. 9L-DsR cells
(4.times.10.sup.4) were transplanted, and 4.times.10.sup.5 of
MSC-EGFP were administered into the tumor or to the contralateral
hemisphere three days after the inoculation of the tumor. The rats
were euthanized 14 days after tumor inoculation and their brains
were excised. (a) and (b) are micrographs of a brain preparation
where MSC-EGFP were administered into the tumor. (c) and (d) are
micrographs of a brain preparation where MSC-EGFP were administered
to the contralateral hemisphere. (a) and (c) are H-E stained, and
(b) and (d) are immunohistochemically stained using an anti-GFP
monoclonal antibody. (e) to (h) are fluorescent micrographs of the
brain where MSC-EGFP were administered into the tumor. (e) shows a
boundary zone between glioma and normal parenchyma. (f) shows the
inside of the tumor, and (g) shows a terminal microsatellite. (h)
is a fluorescent micrograph of a boundary zone between tumor and
normal parenchyma where MSC-EGFP were administered to the
contralateral hemisphere.
[0210] FIG. 36 shows graphs indicating the effects of IL2
genetically modified MSCs on surviving rats to which 9L cells were
inoculated. Survivorship was analyzed using a log-rank test based
on the Kaplan-Meier method. (a) shows the viabilities of rats, with
or without MSC inoculation, after inoculation of 9L cells. (b)
shows the viabilities of rats, with or without inoculation of MSC,
into the tumor three days after tumor inoculation.
[0211] FIG. 37 shows photographs of representative MRIs
(Gd-DTPA-enhanced T1-weighted coronal images). 9L glioma were
inoculated, or not inoculated, with MSCs three days after tumor
inoculation. All animals were subjected to magnetic resonance
imaging analysis every seven days. The tumor volume (mm.sup.3) was
calculated as the sum of image thickness and the area (mm.sup.2) of
Gd-DTPA-enhanced portions in each imaged region.
[0212] FIG. 38 shows photographs indicating the results of
histological analysis of glioma administered with genetically
modified MSCs. Glioma inoculated with unmodified MSCs (a and b) or
with MSC-IL2s (c and d) were histologically analyzed using
hematoxylin and eosin staining. The invasion of CD4-positive
lymphocytes in glioma after inoculation with unmodified MSCs (e) or
MSC-IL2s (f) was detected using a monoclonal antibody W3/25. The
invasion of CD8-positive lymphocytes in glioma after inoculation of
unmodified MSCs (g) or MSC-IL2s (h) was detected using a monoclonal
antibody OX-8.
[0213] FIG. 39 shows graphs indicating the results of investigating
the production of BDNF, GDNF, CNTF, and NT3 by MSCs introduced with
the BDNF, GDNF, CNTF, and NT3 genes. The y-axis indicates cytokine
production (ng/l 05 cell/48-hr), and the x-axis indicates the
multiplicity of infection (pu/cell).
[0214] FIG. 40 is a graph showing the results of assessing
neurological disorders induced by cerebral ischemia. In addition to
the BDNF gene, the GDNF, CNTF, or NT3 gene was introduced into
MSCs, the resulting cells were transplanted to a cerebral
infarction region, and a limb placement test was conducted. The
y-axis indicates the leg-placement score, and the x-axis indicates
data before MCAO, one day after MCAO (before injection), eight days
after MCAO, and 15 days after MCAO, respectively.
[0215] FIG. 41 is a graph showing the infarct volume (HLV) after
local transplantation treatment of MSC-BDNF and MSC-GDNF. The
y-axis indicates the infarct volume (%), and the x-axis indicates
data two days, seven days, and 14 days after MCAO,
respectively.
[0216] FIG. 42 shows photographs of representative T2-weighted
(T2W) images of rats after local administration of DMEM, MSC-BDNF,
MSC-GDNF, MSC-CNTF, or MSC-NT3, taken two days and seven days after
MCAO.
[0217] FIG. 43 shows photographs of MRI images of the group
intravenously administered with MSC-BDNF, the group intravenously
administered with MSC, and an untreated group (control), taken 24
hours, 72 hours, and seven days after MCAO.
[0218] FIG. 44 is a graph showing changes in cerebral infarct
volume after MCAO of the group intravenously administered with
MSC-BDNF, the group intravenously administered with MSC, and an
untreated group (control). The y-axis indicates the infarct volume,
and the x-axis indicates data six hours, 24 hours, 72 hours, and
seven days after MCAO, respectively.
[0219] FIG. 45 is a graph showing treadmill test results of the
group intravenously administered with MSC-BDNF, the group
intravenously administered with MSC, and an untreated group
(control) after MCAO. The y-axis indicates the hignest running
speed, and the x-axis indicates data 24 hours, 72 hours, and seven
days after MCAO, respectively.
[0220] FIG. 46 shows photographs of DW2 (b=1000) images and
T.sub.2WI images in MRI analysis of the cerebral infarctions of an
untreated group (control) and a group intravenously administered
with MSC-PLGF (administered three hours after MCAO), observed at
three hours, 24 hours, three days, and seven days after MCAO,
respectively.
[0221] FIG. 47 shows graphs indicating the results of quantifying
the volume of a region showing abnormal signals developed after
MCAO, observed in MRI analysis over time. The upper graph shows the
results using DWI images, and the lower graph shows the results
using T.sub.2WI images.
[0222] FIG. 48 shows photographs of the brain tissues of the
untreated group (control) and the group intravenously administered
with MSC-PLGF, which tissues were stained with TTC seven days after
MCAO. The upper photographs show the MSC-PLGF treated group, and
the lower photographs show the untreated group (control).
[0223] FIG. 49 shows photographs of the blood vascular system of a
normal rat visualized by staining with Evans Blue and FITC dextran.
The left photograph shows the results using Evans Blue, and the
right photograph shows the result using FITC dextran.
[0224] FIG. 50 shows photographs of the results of using FITC to
visually compare angiogenesis induction in an MCAO-model rat,
locally injected with the angiopoietin gene using an adenoviral
vector, and in an untreated MCAO-model rat. The left images show
results for the MCAO-model rat which was injected with the gene
(Angiopoietin), and the right images show results for the
MCAO-model rat that was not injected with the gene (control).
[0225] FIG. 51 is a graph showing the results of quantifying the
ipsilateral/contralateral ratio using FITC. In FIG. 51, "ANG"
represents angiopoietin treatment.
[0226] FIG. 52 shows photographs indicating the results of using
Evans Blue staining to visually compare angiogenesis induction in
MCAO-model rats injected, and not injected, with a gene. The left
photograph shows results for the MCAO-model rat injected with the
gene (Angiopoietin), and the right photograph shows results for the
MCAO-model rat not injected with the gene (control).
[0227] FIG. 53 is a graph showing the results of a treadmill test
on an MSC-administered group, in which MSCs were locally
administered in the chronic stage after cerebral infarction, and an
untreated group (control). The y-axis indicates the highest running
speed, and the x-axis indicates the number of days after MSC
administration.
[0228] FIG. 54 shows phase-contrast photomicrograph of May-Giemsa
stained BMSCs (A) and PMSCs (B) at 2 and 4 weeks in culture,
respectively. Flow cytometric analysis of cultured BMSCs (E) and
PMSCs (F) with CD45, CD73, CD90, and CD106 antibodies. Dotted lines
in each panel indicate isotype-matched mouse IgG antibody control
staining. Scale bar=10 .mu.m.
[0229] FIG. 55 shows culture expansion of BMSCs (black) and PMSCs
(open square). The cell numbers of both MSCs were counted at each
week. Error bars represent one SD from the mean. * p<0.05
(n=16).
[0230] FIG. 56 shows transformation from MSCs to nestin-positive
neurospheres. When BMSCs (A) and PMSCs (B) were placed in NPBM with
growth factors and were inhibited to adhere on the culture dish,
the cells formed neurospheres (Scale bar=20 .mu.m). RT-PCR analysis
demonstrated that neurospheres transformed from BMSCs showed
nestin-positivity (E-b), which was negative before transformation
(E-a). Nestin also became positive following transformation of
PMSCs (F-b), which was negative in the primary PMSCs (E-a). C and D
showed control mRNA expression of P-Actin of BMSCs and PMSCs,
respectively.
[0231] FIG. 57 shows Neurofilament expression in differentiated
neurosphere cells. Cells differentiated from neurospheres which had
been transformed from BMSCs (A) or PMSCs (B) showed NF-M positivity
in culture. RT-PCR analysis demonstrated that BMSCs (A) and PMSCs
(B) differentiated from neurospheres showed NF-M positivity (E-b;
F-b), which was negative in neurospheres (E-a; F-a). C and D showed
control mRNA expression of P-Actin of BMSCs and PMSCs,
respectively. Scale bar =10 .mu.m.
[0232] FIG. 58 shows GFAP expression in differentiated neurosphere
cells. Immunocytochemical analysis indicated that BMSCs (A) and
PMSCs (B) differentiated from neurospheres showed GFAP positivity
in culture. RT-PCR analysis demonstrated that cells differentiated
from neurospheres which had been transformed from BMSCs showed the
GFAP positivity (E-b), which was negative in neurospheres (E-a).
GFAP also became positive following differentiation in the PMSCs
group (F-b), which was negative before induction (E-a). C and D
demonstrated the mRNA expression of P-Actin of BMSCs and PMSCs for
control, respectively. Scale bar =10 .mu.m.
[0233] FIG. 59 shows May-Giemsa staining of BMSCs (A) and PMSCs (B)
(scale bar =20 .mu.m). Flow cytometric analysis of surface antigen
expression on BMSCs (C) and PMSCs (D). The cells were immunolabeled
with FITC-conjugated and PE-conjugated monoclonal antibody specific
for the indicated surface antigen. Dead cells were eliminated by
forward and side scatter.
[0234] FIG. 60 shows evaluation of the ischemic lesion volume with
Diffusion Weighted Images (DWI). BMSCs or PMSCs were
intravenously-injected immediately after the initial MRI scanning
(6 hrs after MCAO). Images obtained 6 hrs, 1, 3, and 7 days MCAO in
medium-injected (A1-4), BMSC-treated (B1-4), and PMSC-treated group
(C1-4). Summary of lesion volumes evaluated with DWI in each groups
(D). Scale bar =3 mm. * P <0.05
[0235] FIG. 61 shows evaluation of the ischemic lesion volume with
TB.sub.2BWeighted Images (TB2BWI). BMSCs or PMSCs were
intravenously-injected immediately after the initial MRI scanning
(6 hours after MCAO). Images obtained 6 hrs, 1, 3, and 7 days MCAO
in medium-injected (A1-4), BMSC-treated (B1-4), and PMSC-treated
group (C1-4). Summary of lesion volumes evaluated with TB.sub.2BWI
in each groups (D). Scale bar =3 mm. * P <0.05 FIG. 62 shows TTC
Brain sections slices stained with 2,3,5-triphenyl tetrazolium
chloride (TTC) to visualize the ischemic lesions 7 days after MCAO.
TTC-stained brain slices from medium-injected MCAO model rats (A1),
following BMSC-treated (A2), and PMSC-treated (A3) groups. The
Sections were also stained with hematoxylin and eosin at 7 days
post-MCAO. A1though a larger number of inflammatory cells were
obvious in the lesion without cell transplantation (B 1),
parenchymal brain tissue was greatly preserved in the BMSC-treated
(B2) and PMSC-treated group (B3). Inflammatory cells in the lesion
were shown in insert of B 1. On the other hand, preserved neurons
in the lesion were shown in insert of B2 and B3.
Intravenously-administrated BMSCs and PMSCs accumulated in and
around the ischemic lesion hemisphere. BMSCs and PMSCs were
transfected with the reporter gene LacZ. Transplanted LacZ-positive
MSCs (blue cells) were present in the ischemic lesion (BMSCs: C2;
PMSCs: C3). Brain from control (without LacZ transfcted MSCs
transplantation) injected animals with comparable X-gal staining is
shown in Cl. Confocal images (BMSCs: D2; PMSCs: D3) demonstrating a
large number of LacZ-positive cells in the lesion hemisphere.
Confocal image of non-treated group is shown in Dl. Scale bar =3 mm
(A and C), 40 .mu.m (B), and 50 .mu.m (D).
[0236] FIG. 63 shows region of interest (ROI) for dynamic
susceptibility contrast-enhanced perfusion weighted imaging (PWI)
analysis. PWI analysis was carried out at four regions of interest
(ROI) indicated by the boxed numbered areas on the lesion side of
the brain.
[0237] FIG. 64 shows evaluation of hemodynamic state (rCBF maps)
with Perfusion Weighted Images (PWI). BMSCs or PMSCs were
intravenously-injected immediately after the initial MRI scanning
(6 hours after MCAO). Images obtained 6 hrs, 1, 3, and 7 days MCAO
in medium-injected (A), BMSC-treated (B), and PMSC-treated group
(C). Summary of rCBF evaluated with PWI in each groups (D-G), ROI-
l (D), ROI-2 (E), ROI-3 (F), and ROI-4 (G). rCBF ratio (ischemic
lesion / contralateral lesion) at 6 hrs, 1, 3, and 7 days after
MCAO are summarized in figure D-G. Scale bar =3 mm, * P
<0.05.
[0238] FIG. 65 shows seven days after MCAO, the angiogenesis in
boundary zone was analyzed using a three-dimensional analysis
system. FIG. 7A shows the three-dimensional capillary image with
systemically perfused FITC-dextran in the normal rat brain. The
total volume of the micro vessels in the sampled lesion site
decreased 7 days after MCAO (B), but was greater in the
BMSC-treated group (C) and the PMSC-treated group (D). Scale bar
=100 .mu.m.
[0239] FIG. 66 shows the treadmill stress test demonstrated that
maximum speed at which the rats could run on motor driven treadmill
was faster in the BMSCs and PMSCs rats than control. Velocity is
plotted for three times after MCAO induction.
WORKING EXAMPLES
[0240] The present invention will be illustrated in further detail
with reference to several Examples below, which by no means limit
the scope of the invention.
[0241] [Example 1] Transient middle cerebral artery occlusion model
A rat middle cerebral artery occlusion model was used as a stroke
model. Transient middle cerebral artery occlusion (MCAO) was
induced for 45 minutes using the intravascular occlusion method
(E.Z. Longa, P.R. Weinstein, S. Carlson, R. Cummins, Reversible
middle cerebral artery occlusion without craniectomy in rats,
Stroke 20 (1989) 84-91).
[0242] Adult male Sprague-Dawley rats (n=1 13) weighing 250 to 300
g were anaesthetized with 5% isoflurane, and the anesthesia was
mechanically maintained with 1.5% isoflurane in a gaseous mixture
of 70% N.sub.20 and 30% O2 under artificial ventilation. The rectal
temperature was maintained at 37.degree. C. using an infrared heat
lamp. A cannula was inserted into the left femoral artery during
surgery, for measuring blood pH, P.sup.02, and pCO.sub.2. The tip
of a 20.0 to 22.0 mm long 3-0 surgical suture (Dermalon: Sherwood
Davis & Geck, UK) was rounded by heating near a flame, and was
advanced from the external carotid artery into the lumen of the
internal carotid artery, to thereby occlude the origin of the
middle cerebral artery (MCA). The tip of the surgical suture was
extracted from the internal carotid artery 45 minutes after MCAO,
and reperfusion was conducted.
[0243] The physiological parameters (rectal temperature, blood pH,
P0.sub.2, PCO.sub.2, and blood pressure) of all mice were
maintained within normal ranges during surgery and transplant
treatment, and no statistical difference was found between
experimental groups.
[0244] [Example 2] Preparation of bone marrow cells
[0245] Autologous bone marrow was collected from the femur of MCAO
rats, one and a half hours prior to bone marrow cell
transplant.
[0246] The rats were anaesthetized with ketamine (75 mg/kg) and
xylazine (10 mg/kg; i.p.). A 1 cm incision was made in the skin, a
small hole (2x 3 mm) was punctured in the femur using an air drill,
and 1 ml of bone marrow was aspirated using a 22-gauge needle. The
collected samples were diluted and suspended in a medium containing
2 ml of L- 15 medium and 3 ml of Ficoll (Amersham Biosciences).
After centrifugation at 2,000 rpm for 15 minutes, mononuclear cell
fractions were collected and resuspended in 2 ml serum-free medium
(NPMM: Neural Progenitor Cell Maintenance Medium; Clonetics, San
Diego, CA, USA). Following a second centrifugation (2,000 rpm, 15
minutes), cells were suspended in 1 ml of NPMM.
[0247] [Example 3] Experimental groups
[0248] The experiment was conducted using 11 groups (n=88). Nothing
was administered to the Group 1 (control) rats after MCAO (n=8).
The rats in Groups 2 to 6 were intravenously administered with just
the medium (without donor cell administration), 3, 6, 12, 24, and
72 hours after MCAO (n=8 for each group). The rats in Groups 7 to
11 were intravenously administered with the autologous bone marrow
cells (1.0.times.10.sup.7 cells), 3, 6, 12, 24, and 72 hours after
MCAO (each group n=8). Six rats in each group were used to
calculate the infarct volume, and the others were used for other
histological analyses.
[0249] [Example 4] Intravenous administration of autologous bone
marrow cells to rat cerebral infarction model
[0250] LacZ gene was introduced into the bone marrow cells
(mononuclear cell fraction: MCF) before transplantation to the rat
cerebral infarction model (transient middle cerebral artery
occlusion model).
[0251] Adex 1 CA1acZ adenovirus was used to transduce the LacZ gene
into the bone marrow cells. The details of the construction
procedures are described in another document (I. Nakagawa, M.
Murakami, K. Ijima, S. Chikuma, I. Saito, Y. Kanegae, H. Ishikura,
T. Yoshiki, H. Okamoto, A. Kitabatake, T. Uede, Persistent and
secondary adenovirus-mediated hepatic gene expression using
adenoviral vector containing CTLA4IgG, Hum. Gene Ther. 9 (1998)
1739-1745. Y. Nakamura, H. Wakimoto, J. Abe, Y. Kanegae, I. Saito,
M. Aoyagi, K. Hirakawa, H. Hamada, Adoptive immunotherapy with
murine tumor-specific T lymphocytes engineered to secrete
interleukin 2, Cancer Res. 54 (1994) 5757-5760. M. Takiguchi, M.
Murakami, I. Nakagawa, I. Saito, A. Hashimoto, T. Uede, CTLA4IgG
gene delivery prevents autoantibody production and lupus nephritis
in MRL/lpr mice, Life Sci. 66 (2000) 991-1001.). This adenoviral
vector has an adenovirus serotype-5 genome that lacks the E1A, E1B,
and E3 regions to prevent viral replication. Instead of the E1A and
E1B domains, the vector comprises the lacZ gene, which is a
P-galactosidase gene of Escherichia coli. The lacZ gene is
comprised between a CAG promoter, which comprises a cytomegalovirus
enhancer and a chicken P-actin promoter (H. Niwa, K. Yamamura, J.
Miyazaki, Efficient selection for high-expression transfectants
with a novel eukaryotic vector, Gene 108 (1991) 193-199.), and a
rabbit 0-globin polyadenylation signal. This recombinant adenovirus
was propagated in 293 cells and was then isolated. Viral solutions
were stored at -80.degree. C. until use. The autologous bone marrow
cells (1 .0.times.10.sup.7 cells), together with 50 MOI of
AdexlCalacZ, were placed in DMEM containing 10% fetal bovine serum
at 37.degree. C. to allow the adenovirus to infect in vitro.
[0252] The same rats from which bone marrow cells were collected
were then subjected to MCAO. Then, a total volume of 1 ml of a
liquid (NPMM) containing about lx 107 mononuclear cells, just
prepared from the autologous bone marrow, was administered to the
left femoral vein.
[0253] Two weeks after transplantation, cells that expressed
P-galactosidase were detected in vivo.
[0254] First, the brains of the deeply anaesthetized rats were
removed, fixed by leaving to stand in a phosphate buffer to which
0.5% glutaraldehyde had been added. The brain was cut into slices
(100 .mu.m) with a vibratome, and the sections were incubated at
37.degree. C. overnight in a X-Gal developer (phosphate buffered
saline containing 35 mM K.sub.3Fe(CN).sub.6/35 mM
K4Fe(CN).sub.6.3H.sub.2O/2 mM MgCl.sub.2) with X-Gal in a final
concentration of 1 mg/ml. Blue reaction products were formed within
the cells and thus those cells expressing .beta.-galactosidase were
detected.
[0255] The cross section of each brain slice was observed under a
dissecting microscope, and recorded with an image analyzer. The
slices were then fixed by being left to stand overnight in a
phosphate buffer to which 4% paraformaldehyde had been added, being
dehydrated, and then embedded in paraffin. Slices (5 .mu.m) were
cut, and the presence of the blue reaction product (the
.beta.-galactosidase reaction product) was evaluated using a light
microscope (Zeiss: Axioskop FS). Some sections were counter stained
with hematoxylin and eosin.
[0256] X-gal develops a blue color in the host brain tissue, and
thus donor MCF cells were visualized as blue cells in host brain
tissue (FIG. 1). Intravenously administered MCF cells accumulated
in the cerebral infarction area.
[0257] [Example 5] The effect of local and intravenous
administration of MCF on therapeutic effects
[0258] The experimental results show that therapeutic effect
increases with an increasing number of transplanted cells (FIG. 2).
The results also demonstrate that intravenous administration
requires about one hundred times as many cells as local
administration to attain substantially the same therapeutic effect.
Conversely, when one hundred times as many cells are intravenously
administered than locally administered, intravenous administration
can be expected to exhibit substantially the same therapeutic
effect as local administration.
[0259] [Example 6] Therapeutic effects of autologous MCF transplant
on rat cerebral infarction model
[0260] Autologous MCF (1.times.10.sup.7 cells) were transplanted to
the rat cerebral infarction model (transient middle cerebral artery
occlusion model: 45 minutes).
[0261] The extent of infarction lesions was examined using
2,3,5-triphenyltetrazolium chloride (TTC) staining (J.B. Bederson,
L.H. Pitts, S.M. Germano, M.C. Nishimura, R.L. Davis, H.M.
Bartkowski, Evaluation of 2,3,5-triphenyltetrazolium chloride as a
stain for detection and quantification of experimental cerebral
infarction in rats, Stroke 17 (1986) 1304-1308.). Normal brain is
stained red by this method.
[0262] Two weeks after transplantation, the rat was deeply
anaesthetized with sodium pentobarbital (50 mg/kg, i.p.). The brain
was carefully removed and was sliced into 1 mm coronal sections
using a vibratome. Fresh brain sections were immersed for 30
minutes in 37.degree. C. physiological saline containing 2%
2,3,5-triphenyltetrazolium chloride (TTC).
[0263] As a result, the cerebral infarcted area (including both the
cortex and basal ganglia) was slightly stained, and a white image
of the cerebral infarction was clearly visualized in the brain of
the MCAO model rats (FIG. 3).
[0264] The cross-sectional area of infarction in each brain section
was examined with a dissecting microscope, and was measured using
NIH image, which is image analyzing software. Infarct areas in all
brain sections were added, and the total infarct volume of each
brain was calculated.
[0265] The infarct volumes were statistically analyzed. Data are
expressed as "mean+SD".
[0266] Differences between the groups were assessed by ANOVA using
the Scheffes post hoc test for identifying differences between
groups. Differences were deemed statistically significant at
p<0.05.
[0267] Histological analysis of ischemic lesions to which no cells
had been administered (the controls) revealed that ischemic lesions
were found with reproducibility and consistency, and that their
average volume was 258.+-.55 mm.sup.3 (n=6) (FIG. 3F). Of the
occlusion indices used for the infarct model, ischemia as
determined by TTC was highest in the striatum (caudate-putamen),
globus pallidus, and septal nucleus, and was relatively mild in the
cortex.
[0268] Using the same infarction parameters, the bone marrow cells
were intravenously administered 3, 6, 12, 24 and 72 hours after
infarct induction. At all these time points the transplantations
reduced the infarct volume, but better results were obtained when
transplantation was conducted in the early stages after ischemia
induction. When the autologous bone marrow cells were intravenously
administered three hours after MCAO, virtually no infarct was
detected (FIG. 3A); changes in TTC staining were barely detected,
but a slight inflammatory response was detected in the target
infarcted lesion. When the cells were administered six hours after
MCAO, the intensity of TTC staining was reduced in the infarct at
the basal ganglia (40.+-.28 mm.sup.3, n=6) (FIG. 3B). The infarct
gradually increased when the cells were administered 12 hours
(80.+-.25 mm.sup.3, n=6, FIG. 3C), 24 hours (140.+-.18 mm.sup.3,
n=6, FIG. 3D), and 72 hours (180.+-.22 mm.sup.3, n=6, FIG. 3E)
after MCAO.
[0269] The therapeutic effect was more remarkable when the
transplant was conducted earlier.
[0270] However, it is noticeable that a certain degree of
therapeutic effect was obtained even when the treatment was
conducted 72 hours after cerebral infarction.
[0271] The therapeutic effect is considered to be a synergy of the
effects of neuroprotection and neural regeneration. The sooner
after cerebral infarction that the transplant is conducted, the
greater the neuroprotection exhibited. Further, when treatment is
conducted relatively late, the neuroprotective effect is relatively
weak, but the neural regeneration effect becomes stronger
instead.
[0272] The obtained results were quantified and shown in the
histogram of FIG. 4 as the infarct volumes in the control (group
without cell implants), and in the infarction model animals (groups
to which cells were transplanted), which were administered with the
cells 3, 6, 12, 24, and 72 hours after MCAO.
[0273] [Example 7] Effects of intravenous administration of
autologous MCF to rat cerebral infarction model
[0274] Autologous MCFs (1.times.10.sup.7 cells) introduced with
LacZ were intravenously administered to the rat cerebral infarction
model after induction of MCAO. The bone marrow cells were
identified in vivo.
[0275] The phenotype of the transplanted cells in vivo was analyzed
using a laser scanning confocal microscope (n=5). Rats were deeply
anaesthetized with sodium pentobarbital (50 mg/kg, i.p.), and the
heart was perfused first with PBS, then with a fixative solution
containing 4% paraformaldehyde in 0.14 M Sorensen's phosphate
buffer (pH 7.4). The brain was removed, fixed for 24 hours in a
4.degree. C. phosphate buffer containing 4% paraformaldehyde, and
dehydrated in 0.IM PBS solution containing 30% sucrose. The tissue
was placed in O.C.T. compound (Miles Inc.), frozen in liquid
nitrogen, and sliced into 10 .mu.m thick coronal sections using a
cryostat. The sections were dried on silane-coated slide glass.
[0276] To identify the type of cells derived from the donor bone
marrow, a double labeling study was conducted using antibodies
against .DELTA.-galactosidase (polyclonal rabbit
anti-p-galactosidase antibody (IgG) labeled with A1exa Fluor 594,
CHEMICON), neurons (monoclonal mouse anti-neuron-specific enolase
antibody (IgG) labeled with A1exa Fluor 488 [NSE], DAKO) and
astrocytes (monoclonal mouse anti-glial fibrillary acidic protein
antibody (IgG) labeled with A1exa Fluor 488 [GFAP], SIGMA). The
primary antibodies were labeled with A1exa Fluor 488 or A1exa Fluor
594, using a Zenon mouse or rabbit IgG labeling kit (Molecular
Probes) according to the manufacturer's instruction. The tissue
sections were dried on silane-coated slide glass, then washed with
PBS (three times for five minutes), treated for 30 minutes with PBS
containing 0.1 % Triton-X at room temperature, and incubated for
ten minutes with a blocking solution (Protein Block Serum Free,
DAKO) at room temperature. The tissue sections were further reacted
with two types of primary antibodies at room temperature for 60
minutes, then washed with PBS (three times for five minutes). After
immunostaining, the slide glass was covered with a glass cover
using a fluorescence mounting medium (DAKO). A1exa Fluor 488
(green) and A1exa Fluor 594 (red) were excited using a 488 nm laser
beam derived from an argon laser, and a 543 nm laser beam derived
from an He-Ne laser, respectively. Confocal images were obtained
using a laser scanning confocal microscope (Zeiss) and software
(Zeiss).
[0277] The transplanted MCF cells were treated with X-gal to
visualize the donor cells in blue.
[0278] The results showed the transplanted donor cells accumulated
inside and around the cerebral infarction. FIG. 5A shows a coronal
section of the infarcted region comprising accumulated
LacZ-positive cells. Examination with a light microscope indicates
that many cells are present in and around the ischemic lesion (FIG.
5B), and most of these cells were LacZ-positive donor cells (FIG.
5C). Immunohistochemical analysis showed that some of the
LacZ-positive donor cells express NSE, a neuron marker (FIG. 5E) or
GFAP, an astrocyte marker (FIG. 5H). FIGS. 5F and 5I each show
composite images of the LacZ, NSE, and GFAP images. No clear
fluorescence signal was found in the control group. These results
indicate that at least some of transplanted bone marrow cells can
differentiate into neuronal (FIGS. 5E and 5F) and glial cell
lineages (FIGS. 5H and 5I).
[0279] [Example 8] Migration of transplanted MCF cells into the
brain
[0280] The transplanted MCF cells migrated into the brain at a high
rate (FIG. 6). This migration varies with transplant time after
cerebral infarction. For example, in cases where the cells were
intravenously administered three hours after MCAO and infarct
volume was reduced (FIG. 6A), LacZ-positive cells were observed
both in the blood vessel tissue and in the parenchymal brain tissue
of the protected lesion, indicating that the transplanted cells
migrated to sites that would undergo cerebral infarction and be
irreversibly damaged unless treated, and these cells exhibited
remarkable neuroprotective effects, saving nervous system cells
which would ordinarily have been killed (FIGS. 6D and G). When
autologous bone marrow cells were administered 12 hours after MCAO
(FIG. 6B), the pathophysiological features were more complex. A
relatively large number of blue donor cells were found in areas
thought to be severely damaged due to ischemic stress, but a
smaller number of donor cells were present in non-damaged regions
of the lesions (FIGS. 6E and 6H). In addition to the
neuroprotective effect that was observed above, a neural
regenerative effect was also found (FIGS. 6A, 6D, and 6G). In
contrast, when the autologous bone marrow cells were administered
72 hours after MCAO, ischemic damage was much greater (FIG. 6C),
and fewer transplanted cells were found in the lesions (FIGS. 6F
and I). The neuroprotective effect observed above (FIGS. 6A, 6D,
and 6G) was relatively small; however, a strong neural regenerative
effect was observed. It should be noted, however, that even in this
group TTC assays showed that cerebral infarction was suppressed by
bone marrow cell transplantation.
[0281] [Example 9] Confirmation of therapeutic effects of MCF
transplantation by ethological examination
[0282] The therapeutic effects of MCF transplantation were verified
by two ethological examinations: a Morris water maze test to
evaluate learning and memory behaviors, and a treadmill stress test
to evaluate motor function.
[0283] Higher brain finctions (memory, learning) were studied by a
modified water maze test (n=10) based on Morris's method (R.G.M.
Morris, Spatial localization does not depend upon the presence of
local cues, Learn Motiv. 12 (1981) 239-260.). Intravenous
administration of. autologous bone marrow or sham administration
was conducted 12 hours after infarction induction.
[0284] The device comprised a white steel tank, 1.3 m in diameter
and filled with water until 30 cm deep. The water was opacified
with white tempera paint, and was held at 24.degree. C. The walls
of the space comprised visual cues, and these remained in the same
positions during the experiment. In every training trial, a round
ceramic platform 8 cm in diameter was placed 2.5 cm from the water
surface in one quadrant of the tank. On Day 1 of training, a single
habituation trial was conducted by placing each rat on the hidden
platform for 60 seconds. If a rat fell or jumped from the platform,
the rat was saved from the water and returned to the platform.
Quadrant search and swimming speed were monitored with a video
camera mounted to the ceiling and connected to a computer tracked
image analysis system.
[0285] Treadmill stress tests were also conducted. Intravenous
administration of the autologous bone marrow or sham administration
was conducted 12 hours after infarction induction.
[0286] Rats were trained by making them run at a speed of 20 m/min
on a motor-driven treadmill with a slope of 0.degree. for 20
minutes per day, two days a week. The rats were placed on a moving
belt that faced away from an electrified grid, and the rats were
made to run in a direction opposite to that of the belt's movement.
Namely, the rats need to run forwards to avoid a shock (intensity
1.0 mA) to the paw. Only those rats which had learned to avoid the
weak electric shock were included in the test (n=10). The maximum
speeds of the rats running on the motor-driven treadmill were
recorded.
[0287] The behavioral scores recorded in the Morris water maze test
and the treadmill stress test were statistically analyzed. Data are
expressed as "mean.+-.SD". Differences between the groups were
evaluated by ANOVA using Scheffe's post hoc test for identifying
differences between groups. The difference was deemed statistically
significant at p<0.05.
[0288] The experimental data show that improvements in behavior
were observed in both tests (each n=10) (FIGS. 7A and 7B). No
dyskinesis was apparent in normal time observation of both the
untransplanted group and the transplanted group. However, the
treadmill test revealed that the treated rats had higher running
speeds on the motor-driven treadmill than the untreated rats (FIG.
7B). This reveals that transplantation markedly improved the motor
function deterioration due to cerebral infarction. Severe
dyskinesis can potentially affect swimming speed, but the mild
hypokinesis of the present invention is not thought to lead to poor
performance in the Morris water maze test.
[0289] [Example 10] Chronological MRI analysis of therapeutic
effects
[0290] MRI was used to chronologically examine the therapeutic
effects on living animals. This method is used in clinical
examinations and treatments, and data obtained by this method can
be clinically applied without modification, and are very
useful.
[0291] Initially, rats were anaesthetized with ketamine (75 mg/kg)
and xylazine (10 mg/kg, i.p). Each rat was placed in an animal
holder/MRI probe apparatus and positioned inside a magnet. The
rat's head was fixed in an imaging coil. A superconducting magnet
(Oxford Magnet Technologies) of 7 Tesla, having an internal
diameter of 18 cm, interfaced to a Biospec I spectrometer (Bruker
Instruments) was used in every MRI determination. T2-weighted
images were obtained from coronal sections 0.5 mm thick using
visual field 3 cm, TR=3000 ms, TE=30 ms, and reconstructed using a
128 x 128 image matrix.
[0292] Rats in which a cerebral infarction had been induced were
examined using MRI, and abnormal signals were detected from about
three hours after the cerebral infarction. Specifically, a cerebral
ischemic region was detected as a High Intensity Area (HIA) in MRI
(T.sub.2WI) (FIG. 8, upper row). The abnormal signals remained in
the untreated group (FIG. 8, lower row) to eventually form a
cerebral infarcted area.
[0293] [Example 11] Therapeutic effects of using mesenchymal stem
cells The intravenous administration of bone marrow cells
(mononuclear cell fraction: MCF) exhibited significant therapeutic
effects on cerebral infarction. Mesenchymal stem cells (MSCs),
which exist in about 0.1% of MCF, were also used for treatment and
their therapeutic effects were confirmed. Mesenchymal stem cells
can be easily sampled, cultivated, proliferated, and preserved.
[0294] MSCs (1.times.10.sup.7 cells) were administered to the rats
with cerebral infarction of FIG. 8. The cells were intravenously
administered 12 hours after cerebral infarction, and abnormal
signals (HIA), having appeared in MRI tests after cerebral
infarction (FIG. 9, upper row), then disappeared from re-tests one
week after treatment (FIG. 9, lower row).
[0295] Thus, intravenous administration of MSCs was proven to treat
cerebral infarction, which is untreatable at current medical
levels.
[0296] [Example 12] Relationship between number of transplanted MSC
cells and therapeutic effects
[0297] MSCs (1.times.10.sup.4 to 1.times.10.sup.7 cells) were
intravenously administered 12 hours after cerebral infarction.
[0298] To clarify the efficacy of MSCs and hTERT-MSCs transplants
in reducing ischemic lesion volume, cells in different
concentrations (1.times.10.sup.4 to 1.times.10.sup.7 cells) were
intravenously administered 12 hours after infarction induction, and
cerebral images (T2-weighted images) of all tested animals were
obtained 12 hours after MCAO and one week after intravenously
administering different concentrations of hTERT-MSCs. Initial
infarct volumes were estimated using in vivo MRI.
[0299] The left row of FIG. lO(A1-E1) shows simple cerebral images
obtained from five rats, 12 hours after injury. These coronal
forebrain sections were obtained at the caudate-putamen complex
level. An ischemic damaged site is seen as a high-intensity region.
The contralateral brain tissue shows normal signals, enabling
comparison.
[0300] The infarct volume (mm.sup.3) was evaluated by analyzing the
high-intensity regions in a series of images collected from the
entire cerebrum. Estimated infarct volumes were constant among the
tested animals (214.+-.23 mm.sup.3, n=25).
[0301] MRI analysis did not find any change in infarct size when
the vehicle (medium) alone was administered (FIG. 1 OA2). The
infarct volume decreased with an increasing number of intravenously
administered hTERT-MSCs. When 10.sup.4 hTERT-MSCs were
administered, the infarct volume slightly decreased, showing a
slight therapeutic effect (FIG. 10B2) (176.+-.21 mm.sup.3, n=5).
The reduction in infarct volume escalated and the therapeutic
effects became apparent upon administration of 10.sup.5 cells
(138.+-.36 mm.sup.3, n=5) and 10.sup.6 cells (56.+-.18 mm.sup.3,
n=5) (FIGS. 10C2 and 10D2). The infarct volume was most reduced
when 10.sup.7 cells were administered; a virtually complete
therapeutic effect could be expected (FIG. 1 OE2) (23.+-.31
mm.sup.3, n=5). The abnormal signals (HIA) observed before
treatment (FIGS. 1 OA 1, B 1, C1, D1, and E1) remained unchanged if
treatment was not conducted (FIG. 1 OA2), but disappeared partially
or almost completely after treatment (FIGS. 1 OB2, C2, D2, and
E2).
[0302] In another test, primary MSCs were also intravenously
transplanted. Transplantation of 10.sup.6 primary MSCs reduced the
infarct volume to the same extent as in tests using the same number
of hTERT-MSCs (FIG. 1OF) (61.+-.18 mm.sup.3, n=5, vs. 56.+-.18
mm.sup.3, n=5, p=0.69). A supplemental sham control experiment was
conducted using 10.sup.6 dermal fibroblasts (FIG. 1 OF).
Transplantation of 10.sup.6 dermal fibroblasts did not show a
reduction in infarct volume (240.+-.27 mm.sup.3, n=5, p=0.95).
[0303] The therapeutic effect on cerebral infarction of
intravenously administering MSCs correlated with the number of
transplanted cells. Namely, the therapeutic effect was higher when
more cells were transplanted.
[0304] Histological verification was also conducted (FIG. 1).
[0305] After evaluation of lesion volume by MRI analysis, and
before and after cell administration, the rats were perfused,
stained with 2,3,5-triphenyltetrazolium chloride (TTC), and second
independent measurements of infarct volume were obtained. Normal
brain tissues were generally stained with TTC, but the infarction
lesions were not stained or were slightly stained. FIG. 11 A shows
a TTC stain result obtained one week after MCAO without cell
transplantation. Staining on the lesion side was mainly observed in
the corpus-striatum. The lesion volume was calculated by measuring
the region with reduced TTC staining in the forebrain. As with MRI
analysis, infarct size decreased with an increasing number of
transplanted cells. Evaluation by TTC staining showed that
intravenous administration of 10.sup.7 hTERT-MSCs markedly
decreased the lesion volume (FIG. 11B and C).
[0306] [Example 13] Accumulation of transplanted donor cells
[0307] MSCs (1.times.10.sup.6 cells) introduced with LacZ or GFP
were intravenously administered to the rat cerebral infarction
model 12 hours after MCAO.
[0308] The cultured cells were rinsed with phosphate buffered
saline (PBS) and fixed at 4.degree. C. for 15 minutes in a fixative
solution containing 4% paraformaldehyde in 0.14M Sorensen's
phosphate buffer (pH 7.4). The fixed cells were incubated in a
blocking solution containing 0.2% Triton X-100 and 5% normal goat
serum for 15 minutes, then incubated with primary antibodies. The
primary antibodies used were anti-neuron specific enolase (NSE;
1:1000 polyclonal rabbit anti-NSE, Nitirei) antibody, anti-glial
fibrillary acidic protein (GFAP; 1:200 polyclonal rabbit anti-GFAP,
Nitirei) antibody, and anti-Nestin (Nestin; 1:5000 murine
monoclonal anti-Nestin, Chemicon) antibody. For visualizing the
primary antibodies, goat anti-mouse IgG antibody and goat
anti-rabbit IgG antibody with fluorescein (FITC) (1:100, Jackson
ImmunoResearch Laboratories, Inc.), or an alkaline phosphatase
reaction (Zymed) were used according to the manufacturer's
instruction. After immunostaining, a glass cover was placed on a
microscopic slide glass using a mounting medium (Dako) cell-side
down. Photographs were taken using an immunofluorescent microscope
(Axioskop FS; Zeiss).
[0309] The FITC fluorochrome (green) and rhodamine fluorochrome
(red) were excited using a 488 nm laser beam derived from an argon
laser and a 543 nm laser beam derived from a He-Ne laser,
respectively. Confocal images were obtained using a confocal laser
scanning microscope (Zeiss) and software (Zeiss).
[0310] As shown in FIGS. 10 and 11, transplant treatment
significantly reduced infarct volume, however, hTERT-MSCs
expressing GFP were found mainly at the striatum (most infarction
was identified in the untransplanted rats).
[0311] FIGS. 12A and 12B show merged confocal images of GFP
fluorescence and transmitted light image, at low and high power,
respectively, where the images are of the striatum on the infarcted
side. Note the abundance of GFP-positive cellular-like elements.
GFP-expressing cells were mainly concentrated in the
corpus-striatum, but a few were found throughout the affected
hemisphere. No GFP expression was found in images obtained from the
contra lateral striatum, to which infarction had not been
introduced. These data indicate that systemically administered
cells reached the lesion site.
[0312] An immunohistochemical study was conducted to identify
immature neurons (NeuN) and astrocytes (GFAP) in the infarcted
sites of rats transplanted with LacZ-transferred hTERT-MSCs.
[0313] As a result, a small number of NeuN-positive cells and
GFAP-positive cells were co-stained with LacZ (FIGS. 12G to L).
[0314] The transplanted donor cells accumulated in the cerebral
infarction region (FIGS. 12A, B, C, and D). Under a fluorescent
microscope, MSC in host brain tissue becomes green. Donor cells
were not found in the untransplanted group (FIGS. 12E and F). Some
of the donor cells differentiated into neurons (FIGS. 12G, I, and
K) and glial cells (FIGS. 12H, J, and L).
[0315] [Example 14] Confirmation of therapeutic effects of MSC
intravenous administration by metabolic analysis
[0316] The therapeutic effects of intravenous administration of
MSCs on cerebral infarction were examined in terms of
metabolism.
[0317] Specifically, NAA and lactate levels in the brain before and
after cell transplantation were determined using magnetic resonance
spectroscopy (MRS). Correlations have been reported between NAA
signals and the presence of normal neurons, and between an increase
in lactate and neuronal death (Barker, P.B., Gillard, J.H., van
Zijl, P.C., Soher, B.J., Hanley, D.F., Agildere, A.M., Oppenheimer,
S.M. and Bryan, R.N., Acute stroke: evaluation with serial proton
MR spectroscopic imaging, Radiology, 192(3) (1994) 723-32.). MRS
analyses of NAA and lactate levels were conducted in the lesioned
and non-lesioned hemispheres 12 hours after MCAO induction.
[0318] MRS was conducted at TR=1500 msec, TE=20 msec, average
=1024, voxel size 2.5 .times.2.5 .times.2.5 mm.sup.3. The brain was
accurately positioned by holding the rat's head in the flat skull
position, and locating the center of an imaging section 5 mm
posterior to the rhinal fissure.
[0319] Consequently, the therapeutic effects of intravenous
administration of MSCs on cerebral infarction were also verified in
terms of metabolism. Normal hemispheres showed the highest NAA
levels and no lactate signal (FIGS. 13A and D). In contrast, the
lesioned sides showed low NAA levels and high lactate signals
(FIGS. 13A and E). Without cell transplant, NAA signals were low
and lactate signals were high one week after infarction induction
(FIGS. 13B and F). In contrast, after intravenous administration of
10.sup.7 hTERT-MSCs, NAA signals were present, and lactate signals
were low, indicating that the brain tissue was protected by the
transplant treatment (n=5) (FIGS. 13C and G).
[0320] [Example 15] Confirmation of therapeutic effects of MSC
transplantation by ethological analysis
[0321] Two tests were conducted to evaluate the behavioral
abilities of infarction-induced rats and transplanted rats: a
Morris water maze test and a treadmill stress test. These
behavioral tests were started one week after infarction induction,
and were conducted alone or together with cell transplantation.
[0322] The Morris water maze test was conducted every other day.
Control rats learned to mount the platform within several seconds
(Morris, R.G.M., Spatial localization does not depend upon the
presence of local cues., Learn Motiv, 12 (1981) 239-260.). It took
about 140 seconds for MCAO-induced rats to execute the test. Rats
without transplants showed stepwise improvement, and had learned to
mount the platform in about 40 seconds on Day 26 of the test. The
time required for MCAO-induced rats intravenously injected with
hTERT-MSCs to get on the platform gradually decreased, and they
mounted the platform within several seconds by Day 26 of the test,
indicating that transplantation results in remarkable improvement
(FIG. 14A).
[0323] In the treadmill stress test, the maximum treadmill velocity
of control rats (without infarcts) reached about 60 m/min. The
maximum velocity in the treadmill test one week after MCAO
induction alone, or one week after MCAO induction along with
transplantation, was about 35 m/min (FIG. 14B). The untreated rats
showed an increase in treadmill velocity from 11 days after
infarction induction, and gradually improved over 25 days at the
most (46.3.+-.6.1, n=10). The cell-transplanted group showed an
even greater improvement in treadmill velocity, with their maximum
speed 25 days after injury approaching that of the control group
(62.0.+-.7.2, n=10). These results revealed that the transplants
remarkably improve the motor fuiction deterioration due to cerebral
infarction.
[0324] [Example 16] Therapeutic effects of MSC on severe cerebral
infarction
[0325] The therapeutic effects of the regenerative medical
technique using MSCs were studied on rats with severe cerebral
infarction (permanent middle cerebral artery occlusion model), to
determine from what level of tissue damage the treatment can
facilitate recovery. In contrast to the transient middle cerebral
artery occlusion model used in Examples 1 to 15, the model used was
one in which the middle cerebral artery was permanently occluded.
This model was prepared under the same conditions as the model used
in Examples 1 to 15, except for occlusion time.
[0326] Compared to the previously mentioned rat cerebral infarction
model (the transient middle cerebral artery occlusion model: 45
min), those rats with severe cerebral infarction showed a broader
cerebral infarct area (FIG. 15, the portion stained white by TTC
staining).
[0327] Abnormal signals in concordance with cerebral infarction
were also detected in the severe cerebral infarction by MRI
analysis (FIG. 16). Without treatment, the abnormal signals (HIA in
MRI) due to cerebral infarction became more clear with time (12
hours, three days, and seven days after) (FIG. 17).
[0328] MSCs (1.times.10.sup.6 cells) were intravenously
administered to rats with severe cerebral infarction (rat permanent
middle cerebral artery occlusion model). Therapeutic effects were
investigated by MRI analyses (T.sub.2WI) one week after the
cerebral infarction. The cerebral infarction appeared white.
Transplants were timed three hours, six hours, 12 hours, 24 hours,
and 72 hours after cerebral infarction. Cerebral infarction lesions
were clearly observed in the untreated group (uppermost row), but
hardly observed in the group intravenously administered with MSC
three hours after cerebral infarction (FIG. 18). Specifically,
intravenous administration of MSCs also had a remarkable
therapeutic effect on severe cerebral infarction. This therapeutic
effect was more prominent the earlier that transplant was
conducted. It should be noted, however, that some degree of
therapeutic effect is observed even when treatment is conducted 24
hours or more after the cerebral infarction.
[0329] The therapeutic effect is considered to be a synergy of the
effects of neuroprotection and neural regeneration. The sooner
after cerebral infarction that transplant is conducted, the
stronger the neuroprotective and antihydropic actions. When the
transplant is conducted relatively late, the neuroprotection
becomes relatively weak, but the neural regeneration becomes strong
instead.
[0330] FIG. 19 is a graph quantifying results of intravenous
administration of MSCs (1.times.10.sup.6 cells) to severe cerebral
infarctions (rat permanent middle cerebral artery occlusion model),
determined as cerebral infarct volume. The results are divided by
the time elapsed between the disorder onset and MSC administration.
This graph shows that the untreated group had an infarct volume of
about 500 mm.sup.3, but the group treated three hours after MCAO
had an infarct volume of only 200 mm.sup.3, indicating significant
effect. When treatment was conducted within 12 hours of MCAO, the
infarct volume was clearly reduced compared to that in the
untreated group. The earlier the treatment, the greater the
reduction in infarct volume, which means a good prognosis.
[0331] [Example 17] Effects of intravenous administration of MSC on
severe cerebral infarction in hyper acute stage
[0332] The chronological therapeutic effects of intravenous MSC
administration on severe cerebral infarction in the hyper acute
stage were investigated.
[0333] When MSCs (1.times.10.sup.6 cells) were intravenously
administered to severe cerebral infarction three hours after
induction of the cerebral infarction, the abnormal signals (HIAs)
that appeared in MRI tests then disappeared several days into the
treatment, and this effect continued (FIG. 20). The therapeutic
effects of MSCs are exhibited relatively early after
administration, and rather than continuing these effects, the
predominant therapeutic effects of intravenous MSC administration
in the acute stage of cerebral infarction may be neuroprotection
and antihydropic action.
[0334] [Example 18] Effects of intravenous MSC administration on
severe cerebral infarction in the acute stage
[0335] The chronological therapeutic effects of intravenous MSC
administration on severe cerebral infarction in the acute stage
were examined. When MSCs (1.times.10.sup.6 cells) were
intravenously administered to severe cerebral infarction six hours
after the cerebral infarction, the abnormal signals (HIAs) that
appeared in MRI tests immediately before treatment gradually
disappeared after treatment (18 hours, one week, two weeks, and
four weeks after the transplant treatment) (FIG. 21). These
therapeutic effects were not observed in the untreated group (no
data).
[0336] [Example 19] Viability in severe cerebral infarction upon
intravenous MSC administration
[0337] MSCs (1.times.10.sup.6 cells) were intravenously
administered to severe cerebral infarctions (rat permanent middle
cerebral artery occlusion model), and changes in viability after
disorder onset were examined and plotted on a graph.
[0338] The graph demonstrates that treatment by intravenous MSC
administration markedly improved viability in severe cerebral
infarction (rat permanent middle cerebral artery occlusion model)
(FIG. 22). Without treatment, 90% of these same cerebral infarction
model rats died; however, when MSCs (1.times.10.sup.6 cells) were
intravenously administered three hours after cerebral infarction,
80% of the rats survived. This revealed that viability increases
the earlier that treatment is started. Viability after treatment is
also outstanding.
[0339] [Example 20] Effects of MSC transplantation treatment on
clinical symptoms of severe cerebral infarction
[0340] MSC transplant treatment was conducted on severe cerebral
infarction, and clinical symptoms were studied.
[0341] The MSC transplant treatment significant improved clinical
symptoms of severe cerebral infarction (FIG. 23). Treadmill stress
tests demonstrated that transplantation markedly improved motor
ftmction, once deteriorated by cerebral infarction.
[0342] [Example 21] Induction of differentiation of adherent
cultured cells derived from peripheral blood into neural stem cells
or nervous system cells
[0343] Adherent cultured cells such as mesenchymal stem cells were
obtained from the peripheral blood. A large number of these cells
were revealed to be obtainable by hypodermically injecting a factor
such as g-CSF or SCF in advance (FIG. 24).
[0344] The obtained adherent cultured cells could be induced to
differentiate into neural stem cells (Neurosphere). The expression
of Nestin could be verified through RT-PCR (FIG. 25).
[0345] The obtained adherent cultured cells could also be induced
to differentiate into neurons (NF-positive cells) and glial cells
(GFAP-positive cells). The expressions of NF and GFAP could each be
verified by RT-PCR (FIG. 26).
[0346] The following Examples 22 to 31 investigate the therapeutic
effects of transplanting transgenic stem cells to the brain
parenchyma a relatively long time after the onset of cerebral
infarction.
[0347] [Example 22] Preparation of cells Human bone marrow (BM) was
obtained from the posterior iliac crest of healthy adult volunteers
after obtaining their informed consents. This test was approved by
the Institutional Review Board of Sapporo Medical University. BM
mononuclear leukocytes were plated on 150 cm.sup.2 plastic tissue
culture flasks and incubated overnight. After washing away the free
cells, the adherent cells were cultured at 37.degree. C. in
Dulbecco's modified essential medium (DMEM) containing 10%
heat-inactivated fetal bovine serum (FBS) (GIBCO BRL, Rockville,
Maryland) in a humidified atmosphere of 5% CO.sub.2.
[0348] After reaching confluence, the cells were harvested and used
for gene transfection with a retroviral vector, BABE-hygro-hTERT
(Kawano, Y., et al. (2003). Ex vivo expansion of human umbilical
cord hematopoietic progenitor cells using a co-culture system with
human telomerase catalytic subunit (hTERT)-transfected human
stromal cells. Blood 101, 532-540.). MSCs within 40 population
doublings (PD) were used in this study.
[0349] The morphological features of the MSCs were the same as
those previously described by Kobune et al. (Kobune, M., et al.
(2003). Telomerized human multipotent mesenchymal cells can
differentiate into hematopoietic and cobblestone area-supporting
cells. Exp Hematol 31, 715-722.). Adult normal human dermal
fibroblasts (NHDF-Ad) were obtained from TAKARA BIO INC. (Japan)
and were cultured in DMEM containing 10% FBS as described
above.
[0350] [Example 23] Adenoviral vector
[0351] An adenoviral vector (AxCAEGFP-F/RGD) carrying a gene for
RGD-mutated fiber together with a humanized variant of Aequoria
Victoria green fluorescent protein (enhanced GFP: EGFP) under the
control of CA promoter (chicken P-actin promoter with CMV-IE
enhancer) was constructed according to known procedures (Nakamura,
T., Sato, K. and Hamada, H. (2002). Effective gene transfer to
human melanomas via integrin-targeted adenoviral vectors. Hum Gene
Ther 13, 613-626.X Dehari, H., et al. (2003). Enhanced antitumor
effect of RGD fiber-modified adenovirus for gene therapy of oral
cancer. Cancer Gene Ther 10, 75-85.).
[0352] The EGFP gene fragment was isolated from the pEGFP vector
(BD Biosciences Clontech, Palo A1to, CA) and inserted into the
pCAcc vector (PCAEGFP) (Yamauchi, A., et al. (2003).
Pre-administration of angiopoietin-1 followed by VEGF induces
functional and mature vascular formation in a rabbit ischemic
model. J Gene Med in press). The cosmid vector pWEAxCAEGFP-F/RGD so
generated, together with Clal- and EcoT221-digested DNA-TPC from
Ad5dlx-F/RGD, were co-transfected into human embryonic kidney 293
cells. Adenoviral EGFP expression vector, AxCAEGFP-F/RGD, obtained
from isolated plaques, was expanded in these cells and purified by
cesium chloride ultracentrifugation (Kanegae, Y., et al. (1995).
Efficient gene activation in mammalian cells by using recombinant
adenovirus expressing site-specific Cre recombinase. Nucleic Acids
Res 23, 3816-3821.).
[0353] Another adenoviral vector (AxCADsR-F/RGD) carrying a gene
for RGD-mutated fiber together with a humanized Discosoma red
fluorescent protein (DsR) under the control of the CA promoter was
constructed as described above.
[0354] Human BDNF cDNA was cloned by a polymerase chain reaction
using total RNA extracted from primary culture MSCs as the template
(RT-PCR). The identity of the BDNF cDNA obtained in this manner was
verified by sequencing and comparing with the GenBank sequence
XM_006027. The human BDNF primer sequence was: forward
5'-CGGAATTCCACCATGACCATCCTTTTCCTTACTATGGTTA-3' (SEQ ID NO: 1); and
reverse 5'-CCAGATCTATCTTCCCCTTTTAATGGTCAATGTA- 3' (SEQ ID NO:
2).
[0355] A plasmid was obtained by inserting the BDNF cDNA into the
pCAcc vector between the EcoRI site and the Bgl II site, and was
named pCAhBDNF. The plasmid pCAhBDNF was digested by ClaI and the
fragment containing the BDNF cDNA expression unit was isolated by
agarose gel electrophoresis. The adenoviral BDNF expression vector
pWEAxCAhBDNF-F/RGD was prepared using Lipofectamine 2000
(Invitrogen Corporation, Tokyo, Japan).
[0356] Before using the viral vector, the concentration and titer
of the virus was evaluated, and the viral stocks were examined for
potential contamination with replication competent viruses. To
determine viral concentration (particle unit (pu)/ml), the viral
solution was incubated in 0.1 % sodium dodecylsulfate, and
A.sub.260 was measured (Nyberg-Hoffman, C., Shabram, P., Li, W.,
Giroux, D. and Aguilar-Cordova, E. (1997). Sensitivity and
reproducibility in adenoviral infectious titer determination. Nat
Med 3, 808-811.). The viral titers of AxCAhBDNF-F/RGD,
AxCAEGFP-F/RGD, and AxCADsR-F/RGD were 4.35.times.10.sup.11,
5.38.times.10.sup.11, and 1.03.times.10.sup.12 pu/ml,
respectively.
[0357] [Example 24] Adenovirus infection
[0358] Adenovirus-mediated gene transfection was performed as
previously described (Tsuda, H., et al. (2003). Efficient BMP2 gene
transfer and bone formation of mesenchymal stem cells by a
fiber-mutant adenoviral vector. Mol Ther 7, 354-365.).
[0359] Briefly, the cells were seeded onto 15 cm plates at a
density of 2x 10.sup.6 cells per plate. MSCs were exposed to 7.5 ml
of a DMEM suspension containing infectious viral particles at
37.degree. C. for 60 minutes. The cells were infected with
AxCAhBDNF-F/RGD, AxCAEGFP-F/RGD, and AxCADsR-F/RGD at MOIs of
1.times.10.sup.3, 4.times.10.sup.3, and 4.times.10.sup.3 pu/cell,
respectively. The medium was then removed, and the cells washed
with DMEM once, and then re-cultured with normal medium for 24
hours, and transplanted into the brain.
[0360] [Example 25] In vitro detection and quantitative analysis of
immunoreactive human BDNF
[0361] MSC cells transfected with AxCAhBDNF-F/RGD (MSC-BDNF) at
MOIs of 100, 300, 1000, and 3000 pu/cell secreted BDNF at rates of
0.230+0.110, 0.434.+-.0.122, 0.931.+-.0.101, and 1.860.+-.0.410
ng/10.sup.5 cell/48-hr, respectively. Untransfected MSCs also
produced BDNF protein at a rate of 0.0407.+-.0.0059 ng/10.sup.5
cell/48-hr. BDNF production level of MSC-BDNF cells transfected at
an MOI of 1000 pu/cell was 23 times more than in uninfected MSCs
(FIG. 27).
[0362] [Example 26] Transient MCAO animal model and intracerebral
transplantation
[0363] The use of animals in this study was approved by the Animal
Care and Use Committee of Sapporo Medical University, and all
procedures were conducted according to institutional
guidelines.
[0364] Rats were anaesthetized with 3.5% halothane, and were kept
unconscious using a face mask and 1.0% to 2.0% halothane in 70%
N.sub.20 and 30% 02. After surgery, the animals were placed under a
heat lamp to maintain their body temperatures at 37.degree. C.
Local cerebral ischemia was induced in male Wistar rats (each 250
to 300 g) by endovascular middle cerebral artery occlusion (Tamura,
A., Gotoh, 0. and Sano, K. (1986). [Focal cerebral infarction in
the rat: I. Operative technique and physiological monitorings for
chronic model]. No To Shinkei 38, 747-751.). A 5-0 monofilament
nylon suture with a silicone-coated tip was gradually inserted
through an arteriotomy in the right common carotid artery through
the internal carotid artery to a point about 18 mm distal to the
bifurcation of the carotid artery. The nylon suture was extracted
90 minutes into the transitory occlusion, to recover blood flow in
the brain.
[0365] Donor MSCs were transplanted to the brain according to the
method described by Goto et al. (Goto, S., Yamada, K., Yoshikawa,
M., Okamura, A. and Ushio, Y. (1997). GABA receptor agonist
promotes reformation of the striatonigral pathway by transplant
derived from fetal striatal primordia in the lesioned striatum. Exp
Neurol 147, 503-509.).
[0366] After confirming the induction of ischemic brain injury
using the behavioral tests described below, the animals were
randomized for transplantation. The animals were anaesthetized with
intraperitoneal (IP) injection of ketamine (2.7 to 3 mg/100-g) and
xylazine (0.36 to 0.4 mg/100-g) and positioned in a Narishige
stereotaxic frame (Model SR-6N, Narishige Co., Ltd., Japan). Using
a 26-gauge Hamilton syringe, 5 .mu.l of a suspension of
5.times.10.sup.5 MSCs in serum-free DMEM was injected to the right
dorsolateral striatum 4 mm beneath the skull surface and 3 mm
lateral to the bregma level over 2.5 minutes (Paxinos, G., Watson,
C., Pennisi, M. and Topple, A. (1985). Bregma, lambda and the
interaural midpoint in stereotaxic surgery with rats of different
sex, strain and weight. J Neurosci Methods 13, 139-143.). This
position was approximately the ischemic boundary zone. To prevent
rejection of human MSCs transplants, the transplanted rats were
intraperitoneally administered with cyclosporine A (10
mg/kg/day).
[0367] [Example 27] Therapeutic effects of MSC-BDNFs (Experiment
1)
[0368] Experiment 1 was conducted 14 days after MCAO to test the
therapeutic effectiveness of MSC-BDNF. Experimental groups were as
follows: Group 1 (control): Rats in which the ischemic boundary
zone was injected with DMEM 24 hours after MCAO (n=7); Group 2:
Rats in which the ischemic boundary zone was transplanted with
fibroblasts 24 hours after MCAO (NHDF-Ad) (n=6); Group 3: Rats in
which the ischemic boundary zone was injected with MSCs 24 hours
after MCAO (n=7); and Group 4: Rats in which the ischemic boundary
zone was transplanted with MSC-BDNFs 24 hours after MCAO (n=7).
[0369] LPT was performed one, eight, and 15 days after MCAO, and a
treadmill stress test was performed eight and 15 days after MCAO.
MRI was performed on days two, seven, and 14. (1) Limb Placement
Test (LPT) (FIG. 28A) LPTs included eight subtests, described by
Johansson and coworkers (Ohlsson, A. L. and Johansson, B. B.
(1995). Environment influences functional outcome of cerebral
infarction in rats. Stroke 26, 644-649.), and were conducted 24
hours after ischemia induction.
[0370] Briefly, the four limbs of the rats were evaluated using the
top and edges of a counter top. For each subtest, animals were
scored as follows: 0=unable to place their limbs; 1=partial and/or
delayed (by more than 2 seconds) placement of their limbs; and
2=immediate and correct limb placement.
[0371] The neurological scores prior to MCAO were similar for all
animals. One day after MCAO, prior to intracerebral MSC injection,
there was no statistical difference in limb-placement score between
the four ischemic groups. Eight days after MCAO, the
MSC-BDNF-administered rats achieved significantly high
limb-placement scores (8.43.+-.1.52) compared to the control DMEM
rats (3.71.+-.0.49, P=0.0001) and the fibroblast-administered rats
(5.00.+-.1.10, P=0.003). Fifteen days after MCAO, the
MSC-BDNF-administered rats scored 9.14.+-.2.61, which was
significantly higher than the scores seen in the DMEM group
(5.00.+-.1.73, P=0.024). In contrast, on both Day 8 and Day 15 the
MCS-administered rats did not achieve higher scores than the DMEM-
or fibroblast-administered control rats, . (2) Treadmill test (FIG.
28B) In the treadmill test, rats were placed on an accelerating
treadmill (Model MK-680, Muromachi Kikai Co., Ltd., Japan) (Mokry,
J. (1995). Experimental models and behavioural tests used in the
study of Parkinson's disease. Physiol Res 44, 143-150.). The rats
were made to run on a belt, the speed of which gradually increased
by 10 m/s every 10 seconds to a maximum speed of 70 m/s, and were
made to maintain the middle position on that belt. When a rat could
no longer run, the trial was officially ended. The maximum speed at
which each animal could run was measured. The rats were tested
eight and 15 days after MCAO.
[0372] Average treadmill speeds prior to MCAO were comparable
between groups. Eight days after MCAO, the rats of the MSC-BDNF
group achieved significantly higher speeds (23.4.+-.2.6 m/s)
compared to the control DMEM- (9.57.+-.5.6 m/s; P=0.001) and
fibroblast-treated (11.8.+-.6.2 m/s; P=0.017) groups. These
differences were maintained even on Day 15. The MSC-BDNF, control
DMEM, and control fibroblast groups showed speeds of 36.6.+-.9.5,
12.1.+-.9.4 (P=0.002), and 15.8.+-.11.3 (P=0.023), respectively.
MSC-treated rats showed no enhancement in recovery on Day 8 or Day
15. (3) Reduction in infarct volume after MSC-BDNF treatment, as
determined by MRI (FIGS. 29A and 29B) NRI was conducted on all the
animals two, seven, and 14 days after MCAO. The animals were
anaesthetized prior to MRI. The MRI device comprised a
superconductive magnet of 7 T and 18 cm in diameter, connected to a
UNITYINOVA console (Oxford Instruments, UK, and Varian, Inc., Palo
A1to, CA) via an interface. The animals were fixed in the same
position during imaging. Multislice T2-weighted spin echo MR images
(TR 3000 msec, TE 40 msec, field of view 40x 30 mm, section
thickness 2 mm, gapless) were obtained.
[0373] The disposition of the ischemic area was evaluated by
calculating the percent hemisphere lesion volume (% HLV) from the
T2-weighted images using imaging software (Scion Image, Version
Beta 4.0.2, Scion Corporation). Ischemic tissue in each section was
marked, and the infarct volume was calculated considering the
thickness of the section (2 mm/section). To avoid overestimating
infarct volume, a corrected infarct volume (CIV) was calculated
according to the following equation, as described by
Neumann-Haefelin et al. (Neumann-Haefelin, T., et al. (2000).
Serial MRI after transient focal cerebral ischemia in rats:
dynamics of tissue injury, blood-brain barrier damage, and edema
formation. Stroke 31, 1965-1972; discussion 1972-1963.):
CIV=(LT-(RT-RI)).times.d
[0374] In this equation, LT represents the area of the left
hemisphere in mm.sup.2;RT represents the area of the right
hemisphere in mm.sup.2; RI represents the infarcted area in
mm.sup.2; and d represents the thickness of the section (2 mm). The
relative infarct volume (% HLV) is expressed as a percentage of
right hemisphere volume.
[0375] Hyper intensity areas were summed over the six central MR
images in the T2-weighted images, and lesion volume was expressed
as percent contralateral hemisphere lesion volume (% HLV). A1l
groups showed a reduction in % HLV from Day 2 to Day 14. Two days
after MCAO, no significant difference in % HLV was found among the
MSC-BDNF (35.0.+-.4.8%), DMEM (38.7.+-.4.9%), fibroblast
(37.9.+-.3.8%), and MSC (37.8.+-.2.8%) groups, but the % HLV of the
MSC-BDNF group was somewhat reduced compared to the other
groups.
[0376] In contrast, seven days after MCAO, the rats of the MSC-BDNF
group showed a significant reduction in % HLV (25.4.+-.2.8%)
compared to the control DMEM (32.8.+-.4.9%; P=0.002), control
fibroblast- (31.6.+-.2.2%; P=0.015), and control MSC-treated
(30.8.+-.4.3%; P=0.028) groups. After fourteen days, the rats of
the MSC-BDNF group showed a significant reduction in % HLV
(23.7.+-.3.2%) compared to the DMEM control (29.6.+-.3.6%; P=0.01
1). Compared to the control DMEM and fibroblast groups, the
MSC-treated rats did not show any significant recovery in % HLV
seven days (30.8.+-.4.3%) or 14 days (26.2.+-.2.9%) after MCAO.
[0377] [Example 28] In vivo BDNF production level (Experiment 2;
FIG. 30)
[0378] In Experiment 2, growth factors were measured in the
following animal groups: [0379] Group 1 (control): Normal rats
(n=3), [0380] Group 2 (control): DMEM-injected rats (n=3), [0381]
Group 3: MSC-injected rats (n=3), and [0382] Group 4: Rats injected
with MSC-BDNF to the ischemic boundary zone 24 hours after
MCAO.
[0383] The rats were sacrificed seven days after MCAO to measure
BDNF concentration in the local brain tissue.
[0384] The present inventors determined the BDNF level in the local
brain tissue seven days after MCAO using sandwich ELISA.
[0385] MSCs were transfected in vitro at different MOIs (pu/cell),
and culture supernatants were collected 48 hours later for
analysis. Seven days after MCAO, the rats were anaesthetized by the
intraperitoneal administration of ketamine (4.4 to 8 mg/100 g) and
xylazine (1.3 mg/100 g), the brain was removed and sliced while on
ice into coronal sections (200 mg) from -1.0 to 1.0 mm bregma of
the ischemic hemisphere, and these were stored at -80.degree. C.
until use. Each tissue sample was suspended in an equal weight of a
homogenate buffer (1 ml; 137 mM NaCl, 20 mM Tris, 1% NP40, 1 mM
PMSF, 10 .mu.g/ml aprotinin, 1 .mu.g/ml leupeptin, 0.5 mM sodium
vanadate) and homogenized with a Dounce homogenizer. The homogenate
was centrifuged (10,000 g) at 4.degree. C. for ten minutes, and the
supernatant (5 .mu.g/.mu.l) was collected for analysis. The BDNF
concentration of each of the samples (analyzed in triplicate) was
quantified using a commercially available BDNF ELISA kit (Promega,
Madison, Wisconsin).
[0386] The MSC-BDNF-transplanted rats showed significantly
increased BDNF levels in the ischemic hemisphere (45.2.+-.14.8
pg/mg protein) as compared to the DMEM- (12.5.+-.1.9 pg/mg protein;
P=0.0002) or MSC-injected rats (19.3.+-.5.5 pg/mg protein;
P=0.0006). The MSC-treated rats also showed significantly increased
BDNF levels in the ischemic hemisphere as compared to the
DMEM-treated rats (P=0.0124).
[0387] [Example 29] Nuclear DNA fragmentation in MSC-BDNF-treated
animals (Experiments 3A and B; FIG. 31)
[0388] Experiment 3A was conducted to evaluate the intensity of DNA
fragmentation in brain cells seven days after ischemia. The
experimental groups herein are as described in Experiment 2. The
rats were sacrificed seven days after MCAO to evaluate their brain
tissue using TUNEL staining.
[0389] Seven days after MCAO, the rats were anaesthetized and
transcardially perfused, initially, with phosphate-buffered saline
(PBS) and then with PBS containing 4% paraformaldehyde (PFA). The
brains were excised, immersed for two days in PBS containing 4%
PFA, and 30 .mu.m frozen sections (coronal coordinates bregma -1.0
to 1.0 mm) were sliced in a cryostat at -20.degree. C. DNA
fragmentation of cells in the ischemic boundary zone was detected
with an In Situ Apoptosis Detection Kit (Takara Biomedicals, Shiga,
Japan) using the terminal deoxynucleotidyl transferase (dUTP)
nick-end labeling (TUNEL) technique (Gavrieli, Y., Sherman, Y. and
Ben-Sasson, S. A. (1992). Identification of programmed cell death
in situ via specific labeling of nuclear DNA fragmentation. J Cell
Biol 119, 493-501.). Specifically, after protease digestion, the
sections were incubated in a mixture of terminal deoxynucleotidyl
transferase and fluorescein isothiocyanate-labeled dUTP (green).
The sections were then counter-stained with PI (propidium iodide),
which stains red. In these sections the MSCs that were transfected
with AxCADsR-F/RGD were stained red. The total number of positive
red cells was counted in three 1.times.1 mm.sup.2 regions of the
inner boundary zone (Hayashi, T., Abe, K. and Itoyama, Y. (1998).
Reduction of ischemic damage by application of vascular endothelial
growth factor in rat brain after transient ischemia. J Cereb Blood
Flow Metab 18, 887-895.).
[0390] Sections 100 .mu.m thick were prepared using a vibratome and
incubated at 4.degree. C. overnight with a primary antibody diluted
with PBS containing 3% BSA and 0.1% Triton X-100. The primary
antibodies used in this study were anti-neuronal nuclear antigen
(NeuN: mAb377; Chemicon International, Temecula, CA, USA) and
anti-glial fibrillary acidic protein (GFAP: G3893, Sigma)
antibodies. After rinsing in PBS, the sections were incubated at
room temperature for one hour with a fluorescent secondary antibody
(A1exa Fluor 594 goat anti-mouse IgG (H+L): A-111032, Molecular
Probes, Inc.).
[0391] Seven days after MCAO the number of TUNEL-positive cells
(green) in the ischemic boundary zones of MSC-BDNF-injected animals
was significantly less than in the DMEM-injected group (275.+-.73
vs. 55.0.+-.41.0; P=0.013). In contrast, there were significantly
more of these cells in the MSC-injected animals than in the
DMEM-injected animals (173.0.+-.64.9 vs. 55.0.+-.41.0; P=0.20)
(FIGS. 31A, B, and C).
[0392] In Experiment 3B, DNA fragmentation on Day 7 was determined
in animals transplanted with MSC-DsR- (Group 2; n=3) or
MSC-BDNF-DsR- (Group 3; n=3), as well as in control animals (Group
1; n=3).
[0393] A large number of DsR-positive MCS cells were detected less
than 2 mm from the injection site. The MSC-BDNF-treated animals
showed a reduced number of TUNEL-positive transplanted MSCs in the
injection site, as compared to the MSC group (FIG. 31D). In
addition, compared to the MSC group, the MSC-BDNF-treated animals
showed a reduced number of TUNEL-positive cells near MSCs in the
injection site,.
[0394] [Example 30] MSC phenotypes (Experiment 4; FIG. 32)
[0395] Experiment 4 was conducted to determine cell morphology on
Day 7. Experimental groups included DMEM-injected control rats
(Group 1; n=3), MSC-EGFP-transplanted rats (Group 2; n=3), and
MSC-BDNF-EGFP-transplanted rats (Group 3; n=3). Rats were
sacrificed seven days after MCAO to morphologically evaluate brain
tissue.
[0396] To determine whether or not MSCs in the ischemic area
expressed a neuronal phonotype, morphological examinations were
conducted seven days after MCAO. Some transplanted MSCs were
immunopositive to the neuron marker NeuN and astrocyte marker GFAP.
Some displayed fibrous projections, while others had a round shape.
The transplanted MSC-BDNFs showed similar features to those of the
MSCs.
[0397] [Example 31] Data analyses
[0398] The data shown in Examples 22 to 31 are presented as
"means.+-.standard deviation (SD)". The data from the limb
placement and treadmill tests were analyzed using one-way ANOVA and
then Games Howell's post hoc tests. The HLV data were analyzed
using one-way ANOVA and then Tukey's HSD post hoc tests. The ELISA
data were compared between individual groups using Student's
t-tests. TUNEL-positive cell numbers were compared between
individual groups using one-way ANOVA and then Sheffe's post hoc
tests. Significance was assumed if the P value was <0.05.
[0399] The following Examples 32 to 44 examine the therapeutic
effects of transgenic stem cell transplants on brain tumors.
[0400] [Example 32] Establishment of cell lines
[0401] A 9L rat glioma cell line (syngenetic with Fisher 344 rats)
and normal rat kidney (NRK) cells were maintained in Dulbecco's
Modified Eagle's Medium (DMEM, Sigma-A1drich Inc., St Lewis, MO,
USA) supplemented with 10% heat-inactivated fetal bovine serum
(FBS, Invitrogen Life Technologies Inc., Grand Island, NY, USA), 2
mM L-glutamine, 50 .mu.g streptomycin, and 50 units/ml penicillin.
To biologically label the 9L cells, pDsR2-Nl plasmid encoding
humanized Discosoma red fluorescent protein (DsRed2) under the
control of a CMV promoter was purchased from BD Biosciences
Clontech (Palo A1to, CA, USA). Using a DNA complex prepared at a
ratio of 1 .mu.g DNA : 2.5 .mu.l of NeuroPORTER reagent (Gene
Therapy Systems, Inc., San Diego, CA, USA), pDsR2-N1 was
transfected using NeuroPORTER to cells at 50% to 60% confluence. 24
hours after the transfection DsRed2-positive cells were isolated
using FACScalibur (Becton Dickinson Co., Franklin Lakes, NJ, USA),
and further purified by repeating selection 72 hours after the
transfection. The isolated DsRed2-positive 9L cells were selected
in DMEM supplemented with 10% FBS and 1 mg/ml G418 (Invitrogen Life
Technologies) for 14 days to establish a stable cell line
(9L-DsR).
[0402] [Example 33] MSC preparation
[0403] MSCs were prepared from bone marrow according to previously
reported procedures (Tsuda H et al. Efficient BMP2 gene transfer
and bone formation of mesenchymal stem cells by a fiber-mutant
adenoviral vector. Mol Ther 2003; 7: 354-365.).
[0404] Briefly, Fischer 344 rats (nine weeks of age, male) were
sacrificed by cervical dislocation, the femur and tibias were cut
from the soft tissues, and the epiphyses were removed using
rongeurs. The mid shaft bone marrow tissues of the femur and tibias
were then flushed into normal medium (DMEM supplemented with 10%
FBS, 100 unit/ml penicillin, 100 .mu.g/ml streptomycin, 0.25
.mu.g/mi amphotericin-B, and 2 mM L-glutamine). The bone marrow was
successively aspirated into syringes using needles of gradually
decreasing size (18, 20, and 22gauge, respectively) to give a
single cell suspension. The primary culture MSCs were seeded in
normal medium at a density of 5.times.10.sup.7 cells per 10-cm
culture dish. Four days after initial culture, the medium was
replaced with fresh normal medium to remove non-adherent cells.
MSCs were maintained at 37.degree. C. and 5% CO.sub.2, and consumed
medium was exchanged with fresh medium every four days.
[0405] [Example 34] Adenoviral vectors and in vivo gene
transduction
[0406] An adenoviral vector with modified fiber encoding human IL-2
(AxCAhIL2-F/RGD) has been already described (Dehari H et al.
Enhanced antitumor effect of RGD fiber-modified adenovirus for gene
therapy of oral cancer. Cancer Gene Ther 2003; 10: 75-85.). Another
adenoviral vector (AxCAEGFP-F/RGD) with RGD mutated-fiber and a
humanized variant of Aequoria victoria green fluorescent protein
(enhanced GFP: EGFP) under the control of CA promoter (chicken
.DELTA.-actin promoter with CMV-IE enhancer) was constructed as
already described (Dehari H et al. Enhanced antitumor effect of RGD
fiber-modified adenovirus for H. Effective gene transfer to human
melanomas via integrin-targeted adnoviral vectors. Hum Gene Ther
2002; 13: 613-626.).
[0407] An EGFP gene fragment was isolated from the pEGFP vector (BD
BIOSCIENCES CLONTECH, Palo A1to, CA, USA) and inserted into the
pCAcc vector (Yamauchi A et al. Pre-administration of
angiopoietin-1 followed by VEGF induces functional and mature
vascular formation in a rabbit ischemic model. J Gene Med 2003; 5:
994-1004.) (pCAEGFP). An expression cassette containing the EGFP
gene was isolated by restriction enzyme digestion with ClaI, and
inserted into the Clal site of cosmid vector pL. The thus-generated
cosmid vector pLEGFP, together with Clal- and EcoT22I-digested
DNA-TPC derived from Ad5dlx-F/RGD, were co-transfected into human
embryonic kidney 293 cells. Plaques produced from the transfected
293 cells were isolated and evaluated using restriction enzyme
digestion of the viral genome. AxCAEGFP-F/RGD, which is an
adenoviral EGFP expression vector with RGD fiber, obtained from the
isolated plaques, was proliferated in the 293 cells. A1l adenoviral
vectors were proliferated in the 293 cells and purified by cesium
chloride ultracentrifugation. After purification, the virus was
dialyzed against phosphate-buffered saline (PBS) containing 10%
glycerol, and stored at -80.degree. C. Viral titer was determined
in terms of particle units (pu) by spectrophotometry at A.sub.260
nm (Dehari H et al. Enhanced antitumor effect of RGD fiber-modified
adenovirus for gene therapy of oral cancer. Cancer Gene Ther 2003;
10: 75-85.).
[0408] Ex vivo adenoviral gene transduction of primary culture MSCs
has been described. (Tsuda H et al. Efficient BMP2 gene transfer
and bone formation of mesenchymal stem cells by a fiber-mutant
adenoviral vector. Mol Ther 2003; 7: 354-365.).
[0409] Briefly, one day before adenoviral infection, 5x 10.sup.5
MSCs were inoculated on a 10-cm culture dish. The cells were
infected by incubating at 37.degree. C. in 5% CO.sub.2 for one hour
with 5 ml of a preserved viral solution containing either 1000
pu/cell of AxCAEGFP-F/wt or AxCAhIL2-F/RGD. After infection, the
cells were washed twice with PBS (pH 7.4) and supplemented with 10
ml of normal medium.
[0410] [Example 35] Characteristics of primary culture rat MSCs
[0411] The present inventors analyzed the surface antigens on rat
primary culture MSCs using flow cytometry.
[0412] The phenotypes of the primary culture MSCs were analyzed
using FACScalibur (Becton, Dickinson and Company). In summary,
cells were washed twice with PBS containing 0.1% bovine serum
albumin (BSA). After labeling with anti-rat CD73 (SH3), CD45, or
CD1 lb/c monoclonal antibody (Pharmingen, San Diego, CA, USA), the
cells were labeled with a secondary antibody: goat anti-mouse IgG
antibody (Immunotech, Marseille, France) combined with fluorescein
isothiocyanate. Mouse IgG.sub.1-labeled cells (Immunotech) or mouse
IgG2a-labeled cells were analyzed as controls with matching
isotypes.
[0413] The cultured rat MSC cells were CD73 antigen-positive (FIG.
33b). This antigen has been reported as a typical mesenchymal
surface antigens on human MSCs (Kobune M et al. Telomerized human
multipotent mesenchymal cells can differentiate into hematopoietic
and cobblestone area-supporting cells. Exp Hematol 2003; 31:
715-722.).
[0414] No contamination by hematopoietic cells (CD45 or CD11/b) was
detected in the MSC cultures of the present inventors (FIGS. 33c
and 33e).
[0415] [Example 36] The in vitro capacity of MSCs for
differentiation into mesenchymal cells The present inventors
similarly investigated the differentiation of rat MSCs into typical
mesenchymal lineages.
[0416] The in vitro capacities for differentiation of the rat
primary culture MSCs or genetically modified MSCs into typical
mesenchymal lineages were evaluated as previously described (Tsuda
H et al. Efficient BMP2 gene transfer and bone formation of
mesenchymal stem cells by a fiber-mutant adenoviral vector. Mol
Ther 2003; 7: 354-365., Kobune M et al. Telomerized human
multipotent mesenchymal cells can differentiate into hematopoietic
and cobblestone area-supporting cells. Exp Hematol 2003; 31:
715-722.).
[0417] In summary, MSC cells were treated with an osteogenic
differentiation medium supplemented with 80 .mu.g/ml vitamin C
phosphate (Wako Pure Chemical Industries, Ltd., Osaka, Japan), 10
mM sodium .DELTA.-glycerophosphate (CALBIOCHEM, San Diego, CA,
USA), and 10.sup.-7 M dexamethasone (Sigma-A1drich Inc.), or an
adipogenic differentiation medium supplemented with 0.5 .mu.M
hydrocortisone, 500 .mu.M isobutylmethylxanthine, and 60 .mu.M
indomethacin. The differentiation medium was exchanged every three
days until Day 21.
[0418] To confirm osteogenic differentiation, cells were fixed with
10% formalin for ten minutes and stained with 5% silver nitrate
(Sigma-A1drich) for 15 minutes to detect deposition of minerals
(von Kossa staining).
[0419] To detect adipogenic differentiation, the cells were fixed
with 10% formalin for 15 minutes and stained with a fresh Oil Red 0
solution (a 3:2 mixture of 0.5% isopropanol stock solution of Oil
Red 0 and distilled water) to detect lipid droplet formation in
cell cultures.
[0420] The present inventors+ cultured rat MSCs were able to
differentiate into osteocyte lineage (FIG. 33f) and adipocyte
lineage (FIG. 33h). The capacities of primary culture MSCs for
differentiation in to osteogenic and adipogenic lineages were not
affected by LI-2 gene modification using the adenoviral vector
(FIGS. 33g and 33i).
[0421] [Example 37] Effects of MSCs on in vitro proliferation of 9L
cells
[0422] It is unclear whether or not in vivo administration of MSCs
to brain tumors affects tumor growth. However, MSCs are known to
produce cytokines such as fibroblast growth factor (FGF), and other
tumor growth factors (TGFs) capable of supporting tumor growth
(Tille JC, Pepper MS. Mesenchymal cells potentiate vascular
endothelial growth factor-induced angiogenesis in vitro. Exp Cell
Res 2002; 280: 179-191.). The present inventors initially evaluated
the effects of MSC co-culture on the growth of 9L glioma cells in
vitro.
[0423] The present inventors cultured Ds-Red2 (humanized Discosoma
red fluorescent protein)-labeled 9L cells (9L-DsR)
(5.times.10.sup.4 cell/well) alone or with MSC (5.times.10.sup.3
cell/well) or with normal rat kidney (NRK) cells (5.times.10.sup.3
cell/well) in a 6-well plate for 72 hours. The cells were then
trypsinized and counted. The number of 9L-DsR cells was determined
using a flow cytometer (FACScalibur).
[0424] As is shown in FIG. 34a, the proliferation of 9L cells
co-cultured with MSCs (24.5.+-.1.9% inhibition) was significantly
inhibited compared to those co-cultured with NRK cells
(17.4.times.1.9% inhibition, p<0.01).
[0425] To determine the effect of soluble factors released from
MSCs on the proliferation of 9L cells, the present inventors used a
two-chamber culture system.
[0426] MSC or NRK cells were inoculated to DMEM containing 10% FBS
in a Transwell Insert (pore size 0.4 .mu.m, Costar Corporation,
Cambridge, MA, USA) at a density of 1.times.10.sup.5
cell/Transwell. 9L cells were inoculated to DMEM containing 10% FBS
in a well at a density of 5.times.103 cell/well. The co-cultures
were incubated for 72 hours and the cells were directly counted to
determine proliferation in the co-culture system. A1l data are
expressed as percent inhibition, calculated according to the
following equation: Percent Growth Inhibition=[ 1 -(Cell number of
9L-DsR co-cultured with MSC or NRK cells/Cell number of 9L-DsR
cells cultured alone)]x 100.
[0427] As shown in FIG. 34b, significant growth suppression of 9L
cells was also effected by MSCs but was not affected by the NRK
cells cultivated in the different chamber (9.8.+-.3.1% and
1.8.+-.1.2%, respectively, P<0.01).
[0428] These results show that the MSCs themselves have a direct
antitumor effect against 9L glioma cells in vitro, which is
mediated by a soluble factor.
[0429] [Example 38] In vitro migration capability of MSCs
[0430] The present inventors evaluated the migratory nature of MSCs
towards glioma cells in vitro.
[0431] A cell migration assay was conducted using a culture dish
with two chambers: a Transwell (Costar Corporation). Cells were
metabolically labeled with .sup.125I-deoxyuridine (.sup.125U-IUDR,
Amersham Biosciences Corp., Piscataway, NJ, USA).
[0432] In summary, 1.times.105 cell/ml cells were cultured for 24
hours in a medium containing 0.1 .mu.Ci/ml .sup.125I-IUDR. Next,
the cells were washed with DMEM three times and re-suspended in the
same medium. .sup.125I-IUDR-labeled cells (5.times.10.sup.4 cells)
were placed in an upper chamber 8 .mu.m in pore size, and the 9L
cells were placed in a lower chamber. The Transwell was left stand
at 37.degree. C. in 5% CO.sub.2 for 24 hours, and the cells in the
lower chamber were lysed with 1 N NaOH. The radioactivity of the
cellular lysate was assessed using a gamma counter. Results of cell
migration assay are expressed as percentages (count in the lower
chamber as a % of the total cell count).
[0433] Neither MSC nor NRK cells spontaneously migrated, but adding
9L cells to the lower chamber stimulated spontaneous migration
(FIG. 34c). Migration activity increased dose-dependently with an
increasing number of 9L cells. The migration capacity of MSCs was
found to be significantly higher than that of NRK cells
(p<0.01).
[0434] [Example 39] Migration and tumor-tropism of transplanted
MSCs
[0435] Having established the in vitro migration capability of
MSCs, the present inventors investigated whether or not
transplanted MSCs migrate in vivo through a normal brain parenchyma
toward intracranial gliomas.
[0436] To evaluate the intracranial distribution of MSCs,
4.times.10.sup.4 9L-DsR glioma cells were intracranially inoculated
to the right basal glioma, and three days later 4.times.10.sup.5
EGFP (enhanced green fluorescent protein)-labeled MSC (MSC-EGFP)
cells were directly injected into the glioma or into the
contralateral hemisphere. Fourteen days after tumor inoculation,
the rat brain under deep anesthesia was perfused with PBS and then
with 4% paraformaldehyde. The excised brain was fixed with 4%
paraformaldehyde overnight and equilibrated with PBS containing 30%
sucrose for 48 hours. The fixed brain was embedded in OTC compound
(Miles, Inc., Elkhart, IN, USA), snap frozen in liquid nitrogen,
and stored at -70.degree. C. The tissue was cryo-sectioned to 20
.mu.m thick and stained with hematoxylin and eosin (H-E), or
immunohistochemically stained with an anti-GFP monoclonal antibody
(BD Sciences Clontech).
[0437] The sections stained with the first antibody were visualized
using VECTASTATIN Kit (Vector Laboratories, Burlingame, CA, USA).
Imaging was conducted with a Zeiss-Pascal microscope (Carl Zeiss,
Inc., Thornwood, NY, USA).
[0438] As is shown in FIGS. 35a and 35c, a large glioma mass
intensively stained with hematoxylin was found in all rats
inoculated with the 9L cells. The glioma mass occupied the right
hemisphere and caused the midline to be shifted toward the left
hemisphere. Most of the gene-labeled MSCs were observed in the
boundary zone between the tumor and the normal parenchyma, but
after intratumoral injection some of them relatively evenly
infiltrated into the tumor bed (FIG. 35b). MSCs did not migrate
into the distal brain parenchyma or to the contralateral
hemisphere.
[0439] Confocal laser microscopy revealed accumulation of
EGFP-positive MSCs. Most of them maintained their spindle-like
shape at the edge of the DsRed-positive tumor (FIG. 35e).
[0440] The MSCs existed in concordance with glioma cells, which
spread from the main tumor (FIG. 35g). In contrast, MSCs inoculated
into the contralateral hemisphere migrated away from the initial
injection site along the corpus callosum towards the glioma cells
(FIG. 35d). Most of these MSCs remained in the corpus callosum and
at the edge of the adjacent tumor (FIG. 35h).
[0441] MSCs also infiltrated the tumor. Having confirmed the
excellent migration capacity and glioma-tropism of MSCs after
intracranial transplantation, therapeutic genetically modified
cells for treating experimental glioma were therefore prepared in
subsequent steps.
[0442] [Example 40] Human IL-2 production by genetically modified
MSCs
[0443] The human IL-2 (hIL-2) was selected as a therapeutic gene
since the antitumor effects of IL-2 on 9L glioma cells has been
sufficiently established in rat models (Rhines LD et al. Local
immunotherapy with interleukin-2 delivered from biodegradable
polymer microspheres combined with interstitial chemotherapy: a
novel treatment for experimental malignant glioma.
[0444] Neurosurgery 2003; 52: 872-879; discussion 879-880., Iwadate
Y et al. Induction of immunity in peripheral tissues combined with
intracerebral transplantation of interleukin-2-producing cells
eliminates established brain tumors. Cancer Res 2001; 61:
8769-8774.).
[0445] Human IL-2-transfected MSCs (MSC-IL2s) were prepared by
infection with a modified adenoviral vector, as previously
described (Tsuda H et al. Efficient BMP2 gene transfer and bone
formation of mesenchymal stem cells by a fiber-mutant adenoviral
vector. Mol Ther 2003; 7: 354-365.). The rat primary culture MSCs
have low expression levels of adenoviral receptor and
Coxsackie-adenoviral receptor (CAR), and are relatively resistant
to wild type adenoviral infection. A fiber-mutant vector was
therefore used.
[0446] To measure human interleukin-2 (IL-2) production by MSCs
transfected with the human IL-2 gene, MSCs were inoculated to a
24-well plate in triplicate at a density of 10.sup.4 cell/well,
twelve hours prior to adenoviral infection. Next, the cells were
infected with AxCAhIL2-F/RGD and incubated for 72 hours. The
concentration of human IL-2 in the culture supernatant was measured
using ELISA (IL-2 Immunoassay Kit; R&D Systems, Inc.,
Minneapolis, MN, USA).
[0447] A high level of hIL-2 was detected in the supernatant of
MSCs infected with a relatively low concentration of AxCAhIL2-F/RGD
(8.6.+-.0.5 and 24.0.+-.1.7 ng/ml/10.sup.4 cell/72 h at 300 and
1000 particle units/cell, respectively). This agrees with the
present inventor's previous findings. This high-level IL-2
production further increased dose-dependently with increasing
adenoviral concentration.
[0448] [Example 41] Prolonged survival of glioma-bearing rats
transplanted with IL-2 gene-transfected MSCs
[0449] The present inventors investigated whether or not MSC-IL2s
provide in vivo therapeutic benefits.
[0450] Male Fisher 344 rats (seven to eight weeks of age, 200 to
240 g) were purchased from Japan SLC, Inc. (Hamamatsu, Japan). The
animals were anaesthetized and placed in a stereotaxic apparatus
(Narishige Scientific Instrument Lab., Tokyo, Japan). A burr hole
was made at an appropriate location (1 mm posterior to bregma and 3
mm right to midline). A 26-gauge needle was inserted at a position
4 mm ventral from the dura, and 5 .mu.l of a PBS suspension of 9L
tumor cells was inoculated thereto using a 10-.mu.l microsyringe
(Hamilton Company, Reno, NV, USA). Then, 4x 10.sup.4 9L cells were
mixed with 5 .mu.l of a PBS suspension of 4.times.10.sup.5 MSCs or
IL-2-transfected MSCs (MSC-IL2s) (infected with 1000 pu/cell
AxCAhIL2-F/RGD). The resulting cell suspensions were intracranially
injected as described above (FIG. 35a). Injection of the 9L cells
either with unmodified MSCs or EGFP-modified MSCs (MSC-EGFPs) was
also evaluated in the same manner.
[0451] Rats injected with both the 9L glioma cells and MSC-IL2s
showed a significantly prolonged survival (26.3.+-.2.2 days,
P=0.0003 vs. 9L alone, P=0.0008 vs. MSC, P=0.0007 vs. MSC-EGFP)
compared to the control rats, which were injected with 9L alone or
with 9L cells together with unmodified MSCs or MSC-EGFPs (17.11.1,
22.0.+-.0.8, 21.3.+-.1.5 days). The rat groups injected with 9L
cells together with unmodified MSCs (22.0.+-.0.8 days, P=0.0003) or
MSC-EGFPs (21.3.+-.1.5 days, P=0.0003) survived for significantly
longer than the controls, but no significant difference was found
between survival of the MSC group and that of the MSC-EGFP group
(P=0.5881).
[0452] IL-2 gene modification of the MSCs conferred additional
therapeutic advantages to the survival of rats injected with both
MSCs and 9L glioma cells, but genetic modification itself did not
affect their survival.
[0453] The therapeutic benefits of MSC-IL2s were also confirmed in
a therapeutic model of glioma-bearing rats. The rats were
transplanted with 4.times.10.sup.9L glioma cells. On Day 3 after
tumor inoculation, 5 .mu.l of a PBS suspension containing
4.times.10.sup.5 MSCs or MSC-IL2s was transplanted into the tumor
(FIG. 36b).
[0454] The intratumoral inoculation of MSC-IL2s significantly
prolonged survival of 9L glioma-bearing rats (27.7.+-.1.1 days,
P=0.0002 vs. 9L alone) as compared to the control (17.1.+-.1.1
days). The average survival time ofthe MSC-EGFP-injected
glioma-bearing rats (23.2.+-.0.8 days) was significantly less than
that of the MSC-IL2-injected rats (P=0.0024), but significantly
more than the untreated control (P=0.0006).
[0455] [Example 42] Effects of genetically modified MSCs on in vivo
tumor growth evaluated by MRI
[0456] The present inventors evaluated whether or not the prolonged
survival observed after injecting MSC-IL2s or MSCs was related to
tumor growth inhibition. The present inventors conducted magnetic
resonance imaging (MRI) on all animals every seven days to estimate
intracerebral tumor volume.
[0457] The animals were anaesthetized by an intraperitoneal
injection of ketamine (2.7 to 3 mg/100-g) and xylazine (0.36 to 0.4
mg/100-g). Next, 0.2 ml of Gd-DTPA (0.8 to 1.0 mg/kg, Magnevist,
Schering Japan, Tokyo, Japan) was injected to the animals, and
coronal Ti-weighted spin echo images (TR 500 msec, TE 10 msec,
field of view 50.times.50 mm, slice thickness 1.5 mm, gapless) were
obtained using a superconductive magnet of 7 T and 18 cm in
diameter, connected via an interface to UNITYINOVA console (Oxford
Instruments KK, Tokyo, Japan). The tumor volume (mm.sup.3) was
calculated as a sum of the Gd-DTPA enhanced portion of each MRI
imaged area, times the image thickness. The estimated tumor volume
based on MRI has a linear correlation with the actual tumor weight
obtained immediately after the imaging test (Namba H et al.
Evaluation of the bystander effect in experimental brain tumors
bearing herpes simplex virus-thymidine kinase gene by serial
magnetic resonance imaging. Hum Gene Ther 1996; 7: 1847-1852.).
[0458] In TI-weighted imaging, 9L gliomas were clearly recognized
as enhanced regions in coronal cross sections (FIG. 37). As shown
in Table 1 and FIG. 37, 9L gliomas showed progressive growth in the
brains of untreated rats, and reached a fatal volume on Day 14
after tumor inoculation. Table 1 shows the 9L glioma volumes
(mm.sup.3) measured by MRI. TABLE-US-00001 TABLE 1 Day 7.sup.b Day
14 Day 21 Day 28 9L alone 17.6 .+-. 5.8 150.0 .+-. 10.6 ND.sup.c ND
(n = 4) MSCs 5.4 .+-. 1.1* 17.6 .+-. 5.6* 151.3 .+-. 4.8 ND (n = 4)
MSC-IL2s 3.3 .+-. 0.9* 5.3 .+-. 0.9* 16.1 .+-. 0.9** 143.7 .+-. 7.7
(n = 4)
[0459] In Table 1, the symbol "b" represents the number of days
after tumor inoculation; the symbol "c" indicates that the test was
not done; the symbol "*" represents p<0.01 vs. 9L alone; and the
symbol "**" represents p<0.01 vs. MSCs.
[0460] In contrast, the brain tumor volume of MSC-IL2- or
MSC-treated animals was significantly smaller (P<0.01 compared
to the untreated control on Day 14 after tumor inoculation). On Day
14, the unmodified MSC-treated group and the MSC-IL2-treated group
showed no significant difference in tumor volume. However, the IL-2
gene modification showed a clear therapeutic effect 21 days after
tumor inoculation according to MRI. At this time, the gliomas in
unmodified MSC-treated animals had virtually reached fatal volume,
but the tumors remained small when the animals were treated with
MSC-IL2s. These findings on changes in tumor volume agree with the
survival duration in different treatment groups.
[0461] [Example 43] Induction of lymphocyte invasion into gliomas
by MSC-IL2 transplant
[0462] The present inventors investigated whether transplantation
of MSC-IL2s to 9L gliomas induces in vivo immunoreaction.
[0463] To detect the infiltration of CD4 or CD8-positive cells into
gliomas after MSC-IL2 treatment, 4.times.10.sup.4 9L-DsR cells were
transplanted, and 4.times.10.sup.5 cells of MSC-EGFPs or MSC-IL2s
were injected into the tumor three days after tumor inoculation.
The rats were sacrificed seven days after tumor inoculation, and
the excised brains were embedded in paraffin. Brain preparations 6
.mu.m thick were immunohistochemically stained with an anti-rat CD4
(Clone W3/25, Serotec Inc., Oxford, UK) or an anti-rat CD8 (Clone
OX-8, Serotec Inc.) monoclonal antibody, and visualized using a
Vectastain ABC Kit (Vector Laboratories Ltd.).
[0464] Histological analyses of MSC-treated 9L glioma using HE
staining revealed that the IL2 gene-modified MSC-treated 9L gliomas
showed a large amount of mononuclear leukocyte infiltration (FIGS.
38c and d). In contrast, unmodified MSC-transplanted gliomas showed
minimal inflammatory cell infiltration (FIGS. 38a and b). Samples
of the unmodified MSC-transplanted tumors showed virtually no
infiltration of CD4 and CD8 cells (FIGS. 38e and g). In clear
contrast to this, tumors inoculated with IL-2 genetically modified
MSCs showed infiltration of CD4- and CD8-positive lymphocytes
(FIGS. 38f and h).
[0465] [Example 44] Statistical analyses
[0466] Statistical analysis of the cell proliferation assays and
migration assays in Examples 32 to 43 were performed using
Student's t-tests. Scheffe's tests were conducted for tumor volume
assessments on Day 7 and Day 14, and Student's t-tests were
performed on Day 21. P values less than 0.05 in the Student's
t-tests and Scheffe's tests were considered significant.
Statistical analyses of survival were conducted using log-rank
tests.
[0467] [Example 45] MSCs introduced with cytokine genes other than
BDNF
[0468] (1) Cytokine productivity
[0469] Genes other than the BDNF (brain-derived neurotrophic
factor) gene, such as GDNF (glial cell line-derived neurotrophic
factor), CNTF (ciliary neurotrophic factor), or NT3
(neurotrophin-3) gene, were introduced into MSCs, and production of
BDNF, GDNF, CNTF and NT3 by cultivated cells was examined.
[0470] The results are shown in FIG. 39. MSCs transfected with
AxCAhBDNF-F/RGD (MSC-BDNFs) at MOIs of 100, 300, 1000, or 3000
pu/cell secreted BDNF at rates of 0.230.+-.0.110, 0.434.+-.0.122,
0.931.+-.0.101, and 1.860.+-.0.41 ng/10.sup.5 cell/48-hr,
respectively. The untransfected MSCs also produced BDNF
(0.0407.+-.0.0059 ng/10.sup.5 cell/48-hr).
[0471] For GDNF, those MSCs transfected with AxCAhGDNF-F/RGD
(MSC-GDNFs) at MOIs of 300, 1000, or 3000 pu/cell secreted GDNF at
rates of 1.05.+-.0.20, 2.26.+-.0.41, and 4.15.+-.0.54 ng/10.sup.5
cell/48-hr, respectively. Untransfected MSCs also produced GDNF
protein (0.044.+-.0.034 ng/10.sup.5 cell/48-hr).
[0472] For CNTF, MSCs transfected with AxCAhCNT-F/RGD (MS C-CNTFs)
at MOIs of 3000, 1000, or 300 pu/cell secreted CNTF at rates of
0.136.+-.0.028, 0.854.+-.0.145, and 3.58.+-.0.43 ng/10.sup.5
cell/48-hr, respectively. Untransfected MSCs also produced CNTF
protein (0.0520.+-.0.0150 ng/105 cell/48-hr).
[0473] For NT3, MSCs transfected with AxCAhNT3-F/RGD (MSC-NT3s) at
MOIs of 300, 1000, or 3000 pu/cell secreted NT3 at rates of
2.67.+-.0.09, 4.24.+-.0.16, and 6.88.+-.0.07 ng/10.sup.5
cell/48-hr, respectively. Untransfected MSCs also produced NT3
protein (0.12.+-.0.001 ng/10.sup.5 cell/48-hr).
[0474] (2) Evaluation of neurological disorders induced by cerebral
ischemia
[0475] MSC cells introduced with the GDNF, CNTF, or NT3 gene were
transplanted to cerebral infarction regions as in the above
Examples, and limb placement tests were conducted. The limb
placement tests were performed according to the procedure in
Example 27 (1). Limb placement disorder was evaluated according to
the following parameters: 0: severe neurological disorder, 16: no
neurological disorder. Limb placement tests were conducted one
eight, and 15 days after MCAO.
[0476] The results are shown in FIG. 40. The four ischemic groups
showed no statistical difference in limb placement score one day
after MCAO (which was prior to intracranial injection of MSCs).
Eight days after MCAO, the limb placement scores of rats
administered with MSC-BDNF and MSC-GDNF were significantly greater
than those of the DMEM rats (each P<0.05). Fifteen days after
MCAO, the rats administered with MSC-BDNF and MSC-GDNF also showed
significantly higher scores than those in the DMEM group (each
P<0.05).
[0477] In contrast, on both Day 8 and Day 15 the rats administered
with MSC-CNTF and MSC-NT3 did not score higher than the
DMEM-administered control rats. (3) Reduction in infarct volume
after MSC-BDNF and MSC-GDNF treatment as determined by MRI
[0478] MRI was conducted on all animals two, seven, and 14 days
after MCAO. The procedures and evaluations are as in Example 27
(3).
[0479] Compared to the control DMEM group rats, MSC-BDNF group and
MSC-GDNF group rats showed significant reductions in HLV seven days
after MCAO (each P<0.05). Likewise, 14 days after, the MSC-BDNF
group and MSC-GDNF group rats showed significant reductions in HLV
compared to the control DMEM group rats. Both seven and 14 days
after, rats administered with MSC-CNTF or MSC-NT3 showed no
significant recovery in HLV, as compared to the control DMEM group
and the MSC-EGFP group (each P<0.05). The results are shown in
FIG. 41.
[0480] FIG. 42 shows representative T2-weighted (T2W) images of
rats administered with DMEM, MSC-BDNF, MSC-GDNF, MSC-CNTF, or
MSC-NT3, where the images were obtained two and seven days after
MCAO. The MSC-BDNF group and MSC-GDNF group showed a reduction in
ischemic injury volume on Day 7, as compared to other groups.
[0481] [Example 46] Intravenous administration of MSC-BDNF
cells
[0482] MSC cells (10.sup.7 cells) introduced with the BDNF gene
were prepared according to the above Examples. In the rats with
severe cerebral infarction (permanent middle cerebral artery
occlusion model), described in Example 16, cerebral infarction was
produced and the above cells were administered into the left cava
twelve hours later.
[0483] MRI was used to chronologically examine the therapeutic
effects on living animals. The cerebral infarction lesions of the
untreated group (control), MSC-administered group, and
MSC-BDNF-administered group were observed 24 hours, 72 hours, and
seven days after MCAO (FIG. 43).
[0484] In addition, the cerebral infarct volumes of the untreated
group (control), MSC-administered group, and MSC-BDNF-administered
group were calculated and examined six hours, 24 hours, 72 hours,
and seven days after MCAO (FIG. 44).
[0485] A treadmill test was conducted on the untreated group
(control), MSC-administered group, and MSC-BDNF-administered group
24 hours, 72 hours, and seven days after MCAO to examine motion
recovery (FIG. 45).
[0486] A1l data demonstrate that MSCs introduced with the BDNF gene
show higher therapeutic effects than MSCs alone.
[0487] [Example 47] Intravenous administration of MSC-PLGF
cells
[0488] Instead of the BDNF gene, PLGF (placental growth factor) was
introduced into MSCs. Three hours after producing a cerebral
infarction in rats with severe cerebral infarction (permanent
middle cerebral artery occlusion model), described in Example 16,
MSC-PLGF cells (10.sup.7 cells) were administered into the left
cava of the rats.
[0489] The cerebral infarction lesions in the untreated group
(control) and in the MSC-PLGF-administered group were observed
using MRI three hours, 24 hours, three days, and seven days after
MCAO (FIG. 46). The results were compared in DW2 (b=1000) images
and T.sub.2WI images.
[0490] The volumes of areas showing abnormal signals which arose
after MCAO were sequentially quantified using MRI analysis. The
results show that reduction began 24 hours after MCAO in the DWI
images, and three days after MCAO in the T.sub.2WI images (FIG.
47).
[0491] To compare cerebral infarct volumes, the brain tissues of
the untreated group (control) and MSC-PLGF-administered group were
stained with TTC seven days after MCAO (FIG. 48).
[0492] A1l data demonstrate that MSCs introduced with the PLGF gene
show higher therapeutic effects.
[0493] [Example 48] Angiogenetic effects of injecting angiopoietin
gene into a cerebral infarction model
[0494] The angiopoietin gene was directly injected into the
cerebral infarction lesions of a rat cerebral infarction model
(transient middle cerebral artery occlusion model: 45 miutes).
[0495] An adenovirus was used as a vector for introducing the
angiopoietin gene.
[0496] Capillary vessels were visualized using FITC dextran or
Evans Blue to evaluate angiogenesis.
[0497] Images of the blood vascular system of a normal rat
visualized with Evans Blue and FITC dextran are shown in FIG.
49.
[0498] FITC was used to visually compare angiogenesis induction in
MCAO-model rats with or without gene injection (FIG. 50). The
ipsilateral / contralateral ratio was also quantified (FIG.
51).
[0499] Evans Blue was also used to visually compare angiogenesis
induction in MCAO-model rats with or without gene injection (FIG.
52).
[0500] As a result, remarkable angiogenesis was observed. Cerebral
infarction is a disease in which blood vessels are occluded. Thus
incorporating angiogenesis is expected to exhibit remarkable
therapeutic effects.
[0501] [Example 49] Local administration of MSCs in chronic stages
after cerebral infarction
[0502] MSCs were locally administered to rats with severe cerebral
infarction (permanent middle cerebral artery occlusion model),
described in Example 16, in chronic stages after cerebral
infarction, and the therapeutic effect of this was studied.
Specifically, two weeks after MCAO, 1.times.10.sup.4 MSCs were
transplanted into the cerebral infarction region. Treadmill tests
were conducted one day, 14 days, 28 days, and 42 days after MSC
administration, and motor finction recovery was compared with an
MSC-untreated group (the control). As a result, improvements in
motor finction could be seen. The results are shown in FIG. 53.
[0503] Therapeutic effects were fairly low compared to
transplantation in acute stages; however, some therapeutic effects
were still observed. It is preferable to conduct treatment in an
acute stage, since treatment in an acute stage shows greater
therapeutic effect. However, in actual clinics the requirements
from patients already with cerebral infarction are large, and thus
the treatment is also thought to be effective for patients in
chronic stages.
[0504] Accordingly, the agents of the present invention are
preferably used for patients with cranial nerve diseases in an
acute stage, but they are not restricted to acute stages, and are
also effective for patients in chronic stages, for example.
[0505] [Example 51] Cell preparation
[0506] The use of animals in this study was approved by the animal
care and use committee of Sapporo Medical University and all
procedures were carried out in accordance with institutional
guidelines.
[0507] BMSCs
[0508] Bone marrow was obtained from femoral bone in adult female
Sprague-Dawley rats weighing 200-250 g. Rats were anesthetized with
ketamine (50 mg/kg) and xylazine (10mg/kg) i.p. A small hole (2
.times.3 mm) in the femoral bone was made with an air drill
following skin incision (1 cm). Bone marrow (0.5 ml) was aspirated,
diluted to 25 ml with Dulbecco's modified Eagle's medium (DMEM)
(SIGMA, St Louis, MO) supplemented with 10% heat-inactivated fetal
bovine serum (FBS) (GibcoBRL, Grand Island, NY), 2 mM L-glutamine
(Gibco BRL), 100 U/ml penicillin, 0.1 mg/ml streptomycin (Gibco
BRL), was plated on 50-cm.sup.2 Tissue Culture Dish (IWAKI, Tokyo,
Japan), and incubated in a humidified atmosphere of 5 % CO.sub.2 at
37 .degree. C. for three days. BMSCs, when selected by plastic
adhesion, it is preferred to eliminate the nonadherent cells by
replacing the medium about 48 hours after cell seeding.
[0509] When cultures almost reached confluence, the adherent cells
were detached with trypsin-EDTA solution (SIGMA) and subcultured at
1 x 104 cells/ml.
[0510] PMSCs
[0511] Peripheral blood was obtained from adult Sprague-Dawley rats
weighting 200-250 g. Rats were deeply anesthetized with ketamine
(50 mg/kg) and xylazine (10 mg/kg) i.p. Peripheral blood (about 8
ml) was aspirated from vena cava superior with a 18 gauge needle.
Peripheral blood was diluted 1:3 in Puregene RBC Lysis Solution
(Gentra systems, Minneapolis, MN) and was incubated in a 50-ml
conical centrifuge tube for 5 min at room temperature. The tube was
centrifuged at 3500 rpm for 2 minutes and the supernatant was
discarded. The cell pellet was suspended in DMEM supplemented with
10 % FBS, 2 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/ml
streptomycin and was plated on 50-cm.sup.2 plastic tissue culture
dishes and incubated in a humidified atmosphere of 5 % C0.sub.2 at
37 .degree. C. PMSCs, when selected by plastic adhesion, it is
preferred to eliminate the nonadherent cells by replacing the
medium about 48 hours after cell seeding. When cultures almost
reached confluence, the adherent cells were detached with
trypsin-EDTA solution and subcultured at 1 .times.10.sup.4
cells/ml. The cell numbers of both BMSC and PMSC were counted in a
cytometer every a week.
[0512] Some of cultured cells were rinsed in PBS for three times
and fixed for 10 minutes with a fixative solution containing 4 %
paraformaldehyde in 0.14 M Sorensen's phosphate buffer, pH 7.4, at
room temperature. The cells were counterstained with May-Giemsa,
and phase-contrast microphotographs were obtained using a Zeiss
microscope.
[0513] [Example 52] Phenotypic characterization
[0514] Flow cytometric analysis of BMSCs and PMSCs were performed.
Briefly, cell suspensions were washed twice with phosphate-buffered
saline (PBS) containing 0.1 % bovine serum albumin (BSA). For
direct assays fifty thousand cells were incubated with
FITC-conjugated CD 45 (Leukocyte Common Antigen) (BD Bioscience
pharmingen, San Jose, CA), PE-conjugated CD 73
(Ecto-5'-nucleotidase) (BD Bioscience pharmingen), PE-conjugated CD
90 (Thy-1) (eBioscience, San Diego, CA) and PE-conjugated CD
10.sup.6 (VCAM-1) (BD Bioscience pharmingen) at 4 .degree. C. for
30 minutes, and then washed twice with PBS containing 0.1 % BSA.
The cells were analyzed by cytometric analysis using a FACSCalibur
flow cytometer (Becton Dickinson, San Jose, CA) with the use of
CellQuest software.
[0515] [Example 53] Induction of MSCs to floating spheric cells
[0516] When inducing MSCs to floating spheric cells like
neurospheres, MSCs were detached with trypsin-EDTA solution and
were collected in a 50-ml tube in DMEM +10% FBS. After rinsing with
DMEM, cells (5.times.10.sup.4 cells/ml) were suspended in Neural
Progenitor basal medium (NPBM) (Cambrex, One Meadowlands Plaza, NJ)
supplemented with 2 mM L-glutamine, 10 ng/ml epidermal growth
factor (EGF), 10 ng/ml basic fibroblast growth factor (bFGF), 100
U/ml penicillin, 0.1 mg/ml streptomycin, and were plated on
Non-treated dish (IWAKI). Growth factors (EGF and bFGF) were added
every day.
[0517] [Example 54] Differentiation of neurospheres to neural
cells
[0518] When inducing the floating spheric cells (neurospheres) to
neural cells, floating spheric cells were collected by centrifuging
at 1500 rpm for 5 min, suspended in NPBM supplemented with 2 mM
L-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin,
mechanically dissociated, and were plated on plastic tissue culture
dish.
[0519] [Example 55] RT-PCR
[0520] Total RNA was extracted from each cell culture using RNeasy
Mini Kit (QIAGEN, Hilden, Germany). Reverse transcription with 100
ng RNA was performed using the SuperScript II RNase H.sup.- reverse
transcriptase (Invitrogen, Carlsbad, CA). A final volume of 20
.mu.l containing 100 ng RNA, 4 .mu.l First Strand Buffer, 10 mM
dNTPs, 100 mM DTT, 0.5 .mu.g Oligo(dt.sub.12-18 and 100 U of
SuperScript II RNase H.sup.- reverse transcriptase was used. Then a
PCR reaction was carried out using the Hot Star Taq Master Mix Kit
(QIAGEN) in a final volume of 50 .mu.l containing 25 .mu.l Hot Star
Taq Master Mix, and 10 mM upstream sense and downstream sense
primers. Cyclical parameters were denatured at 94 .degree. C. for
30 sec, annealed at 60 .degree. C. for 30 sec, and finally
elongated at 72.degree. C. for 30 sec. Thirty five cycles were
performed for each primer set. PCR products were resolved on 2 %
gel agarose. Primer sequence of amplified products were:
TABLE-US-00002 mouse .beta.-Actin sense
(5'-TGGAATCCTGTGGCATCCATGAAAC-3'), mouse .beta.-Actin antisense
(5'-TAAAACGCAGCTCAGTAACAGTCCG-3'), rat Nestin sense
(5'-CTTAGTCTGGAGGTGGCTACATACA-3'), rat Nestin antisense
(5'-GAGGATAGCAGAAGAACTAGGCACT-3'), rat neurofilament M (NF-M) sense
(5'-GGTCACTTCACATGCCATAGTCAA-3'), rat NF-M antisense
(5'-GGCTCAGTTGGTACTTTGCGTAA-3'), rat glial fibrillary acid protein
(GFAP) sense (5'-ATTCCGCGCCTCTCCCTGTCTC-3'), and rat GFAP
antisense. (5'-GCTTCATCCGCCTCCTGTCTGT-3')
[0521] [Example 56] Immunocytochemical analysis
[0522] To identify the cell type derived from the BMSCs and PMSCs,
immunocytochemical studies were performed with the use of
antibodies to neurons (monoclonal mouse NF-M, SIGMA), and
astrocytes (monoclonal mouse anti-GFAP, SIGMA). Cultured cells were
rinsed in PBS for three times and fixed for 10 minutes with a
fixative solution containing 4 % paraformaldehyde in 0.14 M
Sorensen's phosphate buffer, pH 7.4, at room temperature. After
washing twice in PBS and incubating in PBS containing 0.1 % Triton
X-100 for 10 minutes at room temperature, fixed cells were
incubated for 30 minutes in a blocking solution containing 0.1 %
Triton X-100, and 3% BSA before incubation with the primary
antibody. Primary antibodies are labeled with A1exa Fluor 488 or
A1exa Fluor 594 using Zenon mouse IgG Labeling Kits (Molecular
Probes Inc., Eugene, OR) according to the manufacturer's
instruction. After immunostaining, coverslips were mounted
cell-side down on microscope slides using mounting medium (DAKO
Corp., Carpinteria, CA). Confocal images were obtained using a
Zeiss laser scanning confocal microscope with the use of Zeiss
software.
[0523] All data are presented as mean values.+-.S.D. Differences
among groups were assessed by ANOVA with Scheffe's post hoc test to
identify individual group differences. Differences were deemed
statistically significant at P<0.05.
[0524] [Example 57] Characteristics of BMSCs and PMSCs
[0525] After removing non-adherent cells by replacing the medium
(day 2 in culture), a small portion of attached nucleated cells was
visualized in the BMSC culture dish. By day 14 in culture, the
attached BMSCs had developed into an adherent layer containing
abundant dispersed fibroblast-like cells, and each colony was
predominantly formed by several fibroblast-like cells (FIG. 54A).
By day 28 in culture, the BMSCs had proliferated and tended to form
a near continuous layer comprising mainly fibroblast-like cells
(FIG. 54B).
[0526] In the cultures of PMSCs derived from peripheral blood,
fibroblast-like cells with thin elongated processes around a
central nucleus made their appearance at two weeks after culture
initiation (FIG. 54C). By day 28 in culture, the cells also
continued proliferating and formed a layer of flattened cells (FIG.
54D), with morphological features resembling those of BMSCs.
[0527] FIG. 54E and 54F are flow cytometric data of the expression
of surface antigens on BMSCs and PMSCs, respectively. These results
show that both BMSCs and PMSCs express a similar pattern of surface
antigens: CD45.sup.-, CD73.sup.+, CD90.sup.+, and CD106.sup.-.
[0528] [Example 58] Growth rate
[0529] The number of BMSCs and PMSCs were counted at weekly
intervals in order to characterize the proliferation rate (FIG.
55). BMSCs slowly proliferated in the initial two weeks, and
entered a rapid growth phase for the next four weeks. Proliferation
of BMSCs became slower after 6 weeks, but cell number was
maintained for the next two weeks. The number of BMSCs increased
more than 4 logs for cultures maintained for eight weeks. In
contrast, PMSCs displayed slow but constant growth over 8 weeks in
culture, and expanded over 6-fold.
[0530] [Example 59] Transformation of MCSs to neurospheres
[0531] BMSCs transformed to nestin-positive neurospheres using an
induction protocol (FIG. 56A) described in Methods. BMSCs began
forming floating cell masses and nestin-positivity when they were
inhibited from adhering to the culture dishes (non-treated dishes)
and maintained in the appropriate medium and growth factors (see
Methods). RT-PCR analysis for nestin mRNA expression in cDNA
samples of cultured adherent BMSCs (FIG. 56E-a) and floating
spheric cells (neurospheres) (FIG. 56E-b) are shown in FIG. 3E. The
floating spheric cells displayed an amplification of a PCR fragment
of the expected size for nestin (420-430 bp), but the cultured
non-transformed BMSCs did not (FIG. 56F-a). PMSCs also showed
similar transformation to nestin-positive neurospheres after
induction (FIG. 56B), which was confirmed by RT-PCR (FIG.
56F-b).
[0532] [Example 60] Differentiation from MSC-derived neurosphere to
neural cells
[0533] MSC-derived neurospheres differentiated into neuron- and
glia-like cells in the appropriate culture condition. BMSC-derived
neurospheres differentiated into adherent neural cells when they
were mechanically dissociated, plated on plastic culture dish, and
maintained in NPBM without growth factors. Adherent single layers
contained abundant neuron- and glial-like cells. Immunocytochemical
analysis indicated that the neuronal cells showed NF-M positivity
(FIG. 57A), which was confirmed by RT-PCR. A sample of adherent
cells displayed an amplification of a PCR fragment of the expected
size for NF-M (330-340 bp) (FIG. 57E-b). In addition, GFAP-positive
cell differentiation was also demonstrated with immunostaining
(FIG. 58A) and RT-PCR analysis (FIG. 5E-b). The expected size for
an amplified PCR fragment of GFAP is 430-440 bp. PMSC-derived
neuropshere showed similar differentiating potential to
NF-M-positive neurons (FIG. 57B) and GFAP-positive glia (FIG. 58B),
which were confirmed by RT-PCR in FIG. 57F-b and 58F-b,
respectively.
[0534] [Example 61] Preparation of mesenchymal stem cell prepared
from rat bone marrow
[0535] The use of animals in this study were approved by the animal
care and use committee of Sapporo Medical University and all
procedures were carried out in accordance with institutional
guidelines. Bone marrow was obtained from the femoral bones of the
adult Sprague-Dawley rats weighting 200 - 250 g. Rats were
anesthetized with ketamine (50 mg/kg) and xylazine (10 mg/kg) i.p.
A small hole (2.times.3 mm) in the femoral bone was made with an
air drill following skin incision (1 cm), and 0.5 ml bone marrow
was aspirated with an 18 gauge needle. Bone marrow (0.5 ml) was
mixed with 10 ml of Dulbecco's Modified Eagle Medium (DMEM, Sigma,
USA) +10 %FBS (Gibco, USA) +0.2 mM L-glutamine (Sigma, USA)
+Penicillin / Streptomycin (Sigma, USA) solution, were plated in
100-cm.sup.2 plastic tissue culture flasks and incubated for three
days. After washing away the free cells, the adherent cells were
cultured in the same medium in a humidified atmosphere of 5 %
C0.sub.2 at 37 .degree. C. After reaching confluence, they were
harvested and cryopreserved as primary BMSCs.
[0536] [Example 62] Preparation of mesenchymal stem cells derived
from rat peripheral blood
[0537] Peripheral blood was obtained from the adult Sprague-Dawley
rats weighting 200- 250 g. Rats were anesthetized with ketamine (50
mg/kg) and xylazine (10 mg/kg) i.p. Peripheral blood (7 - 10 ml)
was aspirated from vena cava superior with an 18 gauge needle. The
peripheral blood was mixed with 30 ml of RBC lysis solution (Gentra
systems, Minneapolis, USA), was reacted for 5 minutes at room
temparature, and was centrifuged at 3500 rpm for 2 minutes. The RBC
lysate supernatant was poured off, and the mononuclear cell
fraction was resuspended with DMEM +10 % FBS +0.2 mM L-glutamine
+Penicillin / Streptomycin solution. Cells were plated in
100-cm.sup.2 plastic tissue culture flasks and the adherent cells
were cultured in the same medium in a humidified atmosphere of 5 %
C0.sub.2 at 37 .degree. C. After reaching confluence, they were
harvested and cryopreserved as PMSCs.
[0538] [Example 63] Phenotypic characterization of the primary
BMSCs and PMSCs
[0539] Flow cytometric analysis of BMSCs and PMSCs was performed.
Briefly, cell suspensions were washed twice with PBS containing 0.1
% bovine serum albumin (BSA). For direct assays one million cells
were incubated with FITC-conjugated CD45 (Leukocyte Common Antigen)
(BD Bioscience pharmingen, San Jose, CA), and PE-conjugated CD73
(Ecto-5'-nucleotidase) (BD Bioscience pharmingen, San Jose, CA),
PE-CD90 (Thy-1) (eBioscience, San Diego, CA) and PE-CD10.sup.6
(VCAM-1) (BD Bioscience pharmingen, San Jose, CA) at 4 .degree. C.
for 30 minutes, and then washed twice with PBS containing 0.1 %
BSA. The cells were analyzed by cytometric analysis using a
FACSCalibur flow cytometer (Becton Dickinson) with the use of
CellQuest software.
[0540] [Example 64] Cerebral ischemic model
[0541] The rat MCAO model was used as a stroke model. Permanent
MCAO was by using method of intraluminal vascular occlusion as
described by Longa EZ, Weinstein PR, Carlson S, Cummins R.
Reversible middle cerebral artery occlusion without craniectomy in
rats. Stroke 1989;20:84-91. Adult female Sprague-Dawley rats
weighing 250 - 300 g were initially anesthetized with ketamine (50
mg/kg) and xylazine (10 mg/kg) i.p. A length of 20.0 - 22.0 mm 4-0
surgical suture (Dermalon, Sherwood Davis and Geck, UK) with the
tip rounded by heating near a flame was advanced from the external
carotid artery into the lumen of the internal carotid artery until
it blocked the origin of the MCA.
[0542] [Example 65] Transplantation procedures
[0543] Experiments consisted of three groups (n=85). In group 1
(control), rats were given medium alone (without donor cell
administration) injected i.v. at 6 h after MCAO Gust after the
initial MRI measurement) (n=15). In group 2, rats were given rat
BMSCs (1.0 x .sub.10p6P) in 1 ml total fluid volume (DMEM) injected
i.v. at 6 h after MCAO (n=15). In group 3, rats were given rat
PMSCs (1.0.times.10.sup.6) injected i.v. at 6 h after MCAO (n=15).
A1l rats were daily injected with cyclosporine (lOmg /kg) i.p. Five
rats in each group were used to calculate the infarct lesion
volume, and the remaining rats were used for the additional
histological, behavior and other analysis.
[0544] In som e experiments, Adex1CA1acZ adenovirus was used to
transduce the LacZ gene into the MSCs. Details of the construction
procedures are described in the cited references to Iihoshi (Brain
Res 2004;1007:1-9), Nomura (Neuorscience 2005;136:161-169),
Nakamura (Cancer Res 1994;54:5757-5760), Nakagawa (Hum Gene Ther
1998;9:1739-1745), and Takiguchi (Life Sci 2000; 6:991-1001). This
adenoviral vector carries an adenovirus serotype-5 genome lacking
the E1A, E1B and E3 regions to prevent virus replication, and
contains the Escherichia coli h-galactosidase gene, lacZ gene,
between the CAG promoter, composed of the cytomegalovirus enhancer
plus the chicken .DELTA.-actin promoter, and the rabbit
.DELTA.-globin polyadenylation signal in the place of the E1A and
E1B regions. The recombinant adenovirus was propagated and isolated
in 293 cells. Viral solutions were stored at -80 .degree. C. until
use. For in vitro adenoviral infection, 1.0.times.10.sup.6 rat MSCs
were placed with AdexICA1acZ at 50 MOI for lh and incubated at 37
.degree. C. in DMEM containing 10 % fetal calf serum.
[0545] [Example 66] MR Imaging
[0546] Rats were anesthetized with ketamine (50 mg/kg) and xylazine
(10 mg/kg) i.p. The femoral vein of rats was cannulated for
contrast agent injection. Each rat was placed in an animal
holder/MRI probe apparatus and positioned inside the magnet. The
animal's head was held in place inside the imaging coil. A1l MRI
measurements were performed using a 7-T, 18-cm-bore superconducting
magnet (Oxford Magnet Technologies) interfaced to a UNITYINOVA
console (Oxford Instruments, UK and Varian, Inc., Palo A1to, CA,
USA). T.sub.2 weighted images (T.sub.2WI) were obtained from a
1.0-mm-thick coronal section with a 0.5 mm gap using a 30
mm.times.30 mm field of view, TR =3000 ms, TE =37 ms, and
reconstructed using a 256.times.128 image matrix. Diffusion
weighted images (DWI) were obtained at the same condition as
T.sub.2 WI except b value (b value =966) and image matrix
(128.times.128). Accurate positioning of the brain was performed to
center the image slice 5 mm posterior to the rhinal fissure with
the head of the rat held in a flat skull position. MRI measurements
were obtained 6 hours, 1 day, 3 days and 7 days after MCAO.
[0547] The ischemic lesion area was calculated from both T.sub.2WI
and DWI using imaging software (Scion Image, Version Beta 4.0.2,
Scion Corporation), based on the method described by Nomura
(Neuorscience 2005;136:161-169) and Neumann-Haefelin (Stroke
2000;31:1965-1972). For each slice, the higher intensity lesions in
both T.sub.2WI and DWI where the signal intensity were 1.25 times
higher than the counterpart in the contra-lateral brain lesion were
marked as the ischemic lesion area, and infarct volume was
calculated taking slice thickness (1 mm/slice) into account.
[0548] [Example 67] Dynamic susceptibility contrast-enhanced
perfusion weighted imaging (PWI)
[0549] PWI was acquired using T.sub.2 weighted (TR=13 msec, TE=6.0
msec) gradient echo sequence. A dynamic image series of 30
measurements resulted in a total scan time of 26 seconds, with a
FOV of 30 mm, and image acquisition matrix of 128.times.64 which
was interpolated by zero-filling to 512.times.512. During the
dynamic series, a triple dose (0.6 ml/kg) bolus injection of
Magnevist (Schering AG, Deutschland) was started after the .sub.5th
acquired volume to ensure a sufficient pre-contrast baseline.
Images were reconstructed by an Inova Vision. PWI measurements were
obtained 6 hrs, 1, 3 and 7 days after MCAO. For the PWI and
PWI-derived parameter maps, only one representative slice
(involving cortex and stria terminalis) with the maximum lesion
involving both cortex and striatum was chosen for CBF
quantification. The readout of abnormal rCBF from the regions of
perfusion deficiency as a percentage of that measured in the
contralateral brain was generated using Perfusion Solver software.
Regions of interest (ROI) consist of four groups, based on the
results of DWI, TB.sub.2BWI and PWI. ROI-1 is defined as abnormal
in all images, ROI-2 as normal in only TB.sub.2BWI and abnormal in
others, ROI-3 as abnormal in only PWI and normal in others, ROI-4
as normal in all images (FIG. 63).
[0550] [Example 68] Histological analysis
[0551] TTC staining and quantitative analysis of infarct volume
[0552] One week after transplantation, the rats were anesthetized
with ketamine (50 mg/kg) and xylazine (10 mg/kg) i.p. The brains
were removed carefully and dissected into coronal lmm sections
using a vibratome. The fresh brain slices were immersed in a 2 %
solution of 2,3,5-triphenyl tetrazolium chloride (TTC) in normal
saline at 37 .degree. C. for 30 min. The cross-sectional area of
infarction in each brain slice was examined with a dissection
microscope and was measured using an image analysis software (Adobe
Photoshop). The total infarct volume for each brain was calculated
by summation of the infracted area of all brain slices. H-E
staining
[0553] The rats were anesthetized with ketamine (50 mg/kg) and
xylazine (10 mg/kg) i.p. and perfused through the heart, first with
PBS, and then with a fixative solution containing 10 %
paraformaldehyde in 0.14 M Sorensen's phosphate buffer, pH 7.4.
Brains were removed and placed in 10 % paraformaldehyde in
phosphate-buffer overnight, dehydrated, and embedded in paraffin.
Transverse sections (1.5 .mu.m) were cut, and were counterstained
with hematoxylin and eosin.
[0554] [Example 69] Detection of donor MSCs and phenotypic analysis
in vivo X-gal staining
[0555] One week after transplantation, brains of the deeply
anesthetized rats were removed and fixed in 0.5 % glutaraldehyde in
phosphate buffer for 1 h. Brains were removed and brain slices
(1000 .mu.m) were cut with a vibratome and .DELTA.-galactosidase
expressing cells were detected by incubating the sections at 37
.degree. C. overnight with X-gal to a final concentration of 1
mg/ml in X-Gal developer (35 mM K.sub.3Fe(CN).sub.6/ 35 mM
K4Fe(CN).sub.63H.sub.20/ 2 mM MgCI.sub.2 in phosphate-buffered
saline) to form a blue reaction product within the cell.
[0556] [Example 70] Immunohistochemistry
[0557] One week after transplantation, analysis of the transplanted
cells in vivo was carried out using laser scanning confocal
microscopy. Brains of the deeply anesthetized rats were removed,
fixed in 4 % paraformaldehyde in phosphate-buffer, dehydrated with
30 % sucrose in 0.1 M PBS for overnight, and frozen in powdered dry
ice. Coronal cryostat sections (10 .mu.m) were processed for
immunohistochemistry. To identify the cells derived from the donor
peripheral blood, immuno-labeling studies were performed with the
use of antibodies to beta-galactosidase (rhodamine-labeled
polyclonal rabbit anti-beta-galactosidase antibody, DAKO). To
excite the rhodamine fluorochrome (red), a 543-nm laser line from a
HeNe laser was used. Confocal images were obtained using a Zeiss
laser scanning confocal microscope with the use of Zeiss
software.
[0558] [Example 71] Capillary vessels in ischemic brain
[0559] To examine capillary vessels in ischemic brain, fluorescein
isothiocyanate (FITC) dextran (2 .times.I.sup.1P molecular weight,
Sigma; 0.1 mL of 50 mg/mL) was administered intravenously to the
ischemic rats subjected to 7 days of MCAO. Brains were removed and
brain slices (100 .mu.m) were cut with a vibratome. To excite the
FITC (green), a 488-nm laserline generated by an argon laser was
used. Confocal images were obtained using a Zeiss laser scanning
confocal microscope with the use of Zeiss software, and vessel
volumes were measured in the three dimensions using the software of
Zeiss LSM.
[0560] [Example 72] Treadmill stress test
[0561] Rats were trained 20 min per day for 2 days a week to run on
a motor driven treadmill at a speed of 20 m/min. Rats were placed
on a moving belt facing away from the electrified grid and induced
to run in the direction opposite of the movement of the belt. Thus,
to avoid foot-shocks (with intensity in 1.0 mA), the rats had to
move forward. Only the rats that had leaned to avoid the mild
electrical shock were included in this study (n=15). The maximum
speed at which the rats could run on a motor driven treadmill was
recorded.
[0562] The lesion volume, the rCBF ratio, the capillary vascular
volume, and the behavior scores (treadmill stress test) recorded
were statistically analyzed. Data are presented as mean
values.+-.S.D. Differences among groups were assessed by ANOVA with
Scheffe's post hoc test or Kruskal-Wallis test to identify
individual group differences. Differences were deemed statistically
significant at P<0.05.
[0563] [Example 73] Characteristics of BMSCs and PMSCs
[0564] BMSCs and PMSCs cultured as plastic adherent cells could be
maintained in vitro. The morphological features of the BMSCs are
shown in FIG. 59A. Characteristic flattened and spindle-shaped
cells can be recognized. An antigenic characteristic feature of
BMSCs is a CD45 (-), CD73 (+), CD90 (+), CD10.sup.6 (-) cell
surface phenotype (FIG. 59C). The morphological (FIG. 59B) and
antigenic (FIG. 59D) characteristics of PMSCs are very similar to
those of BMSCs.
[0565] [Example 74] Characterization of ischemic lesion size by
magnetic resonance image analysis
[0566] An estimate of lesion size was obtained using in vivo MRI
(see Experimental Procedures). Brain images (DWI and T.sub.2WI)
were collected from all experimental animals 6 hrs, 1, 3 and 7 days
after MCAO. The cells were intravenously delivered immediately
after the 6 hrs MRI. The upper row in FIG. 60A corresponds to 6 hrs
DWI post-MCAO for control (A1), BMSCs (B1) and PMSCs (C1) injected
rats. Respective images are shown at 1, 3 and 7 days for each
group. These coronal forebrain sections were obtained at the level
of caudato-putamen complex. Note the reduction in density in
lesions on the right side of the brains that were subjected to
ischemic injury. Lesion volume (mm.sup.3) was determined by
analysis of high intensity areas on serial images collected through
the cerebrum (see Experimental Procedures).
[0567] At 6 hrs post-MCAO, lesion volume of DWI was similar for the
three groups (FIG. 60D). Lesion volume increased at 1 day, but was
less for both the BMSC and PMSC groups. The control lesion group
showed a reduced lesion volume at 3 and 7 days, but the MSC groups
showed greater reduction in lesion volume (FIG. 60D).
[0568] Using T.sub.2WI (FIG. 61), infarction volume was similar in
the three groups at 6 hrs post-MCAO (FIG. 61D). Both the BMSC and
PMSC injected groups showed reduced lesion volume at 1, 3, and 7
days post MCAO.
[0569] A difference between DWI and TB.sub.2BWI was observed.
Lesion volume decreased after 1 day in the three groups in the DWI
analysis. Using T.sub.2WI, lesion volume increased from 1 to 3
days. However, the BMSC and PMSC groups showed reduced volumes in
both DWI and T.sub.2WI analysis.
[0570] [Example 75] Histological determination of infarction
volume
[0571] After completion of the MRI analysis to estimate lesion
volume, before and after cell delivery, the animals were perfused
and stained with TTC to obtain a second independent measure of
infarction volume. Normal brain (gray matter) tissue typically
stains with TTC, but infracted lesions show no or reduced staining.
TTC-staining that was obtained one week after MCAO without cell
transplantation is shown in FIG. 63A-1. Note the reduced staining
on the lesion side. Lesion volume was calculated by measuring the
area of reduced TTC-staining in the forebrain (see Experimental
Procedures). As with MRI analysis, there was a progressive
reduction in infarction size with both BMSCs and PMSCs treatment
(FIG. 62A-2, 62A-3, respectively). Lesion volume was 263.0.+-.35.26
mmP.sup.3P (control group; n-5), 180.0 .+-.5.89 mmP.sup.3P, (BMSCs
transplantation; n=5), and 185.86.+-.19.12 mmP.sup.3P (PMSCs; n=5,
p<0.05).
[0572] HE stained sections from the sham lesion cortex (FIG.
62B-1), and cortex from BMSCs (FIG. 624B-2) and PMSCs (FIG. 62B-3)
groups indicated more neuron preservation and fewer inflammation
cells were present in the cell infusion groups.
[0573] [Example 76] Identification and characterization of donor
cells in vivo
[0574] LacZ-transfected BMSCs and PMSCs that had been i.v.
administered (1.0.times.10.sup.6 cells) 6 hours after MCAO were
identified in vivo. The LacZ-expressing MSCs were found primarily
in the lesion. The transmitted light images in the LacZ-transfected
BMSCs and PMSCs are shown in FIG. 62C-2 and FIG. 62C-3,
respectively. Note the abundance of LacZ-positive
blue-cellular-like elements in and around the lesion, indicating
that systemic deliver of both types of cells reached the lesion
site. There was a paucity of blue staining in the non-treated group
(FIG. 62C-1). Immunohistochemical studies were carried out to
identify LacZ-positive cells in and around the lesion zone in
animals transplanted with LacZ-transfected MSCs. The micro
photographs of BMSCs (FIG. 62D-2) and PMSCs (FIG. 62D-3)
demonstrated a large number of LacZ-positive cells in and around
the lesion (300.+-.30 cells/mm.sup.2, n=5), although there was
virtually no LacZ-positive cells in the non-damaged hemisphere.
[0575] [Example 77] Dynamic susceptibility contrast-enhanced
PWI
[0576] The PWI-derived parameter maps to assess regional cerebral
blood flow allowed further quantitative analysis for the
hemodynamic changes of the lesions (see Methods). FIG. 64A-C shows
images obtained at 6 hrs (row 1), 1 day (row 2), 3 days (row 3),
and 7 days (row 4). Control, BMSC- and PMSC-injected groups are in
columns A, B and C, respectively.
[0577] The four regions of interest (ROI) for the analysis are
defined in Methods and shown in FIG. 63. The severity of the lesion
was greatest in ROI-1 and progressively less in ROI-2 through
ROI-4. A rCBF ratio was calculated at each ROI from PWI obtained in
the infarction hemisphere divided by that of the non-infarcted
hemisphere. In ROI-1, the rCBF ratio of control, BMSC-treated, and
PMSC-treated groups were similar and decreased to less than 20 % at
6 hours post-MCAO, and remained low at 3 and 7 days (FIG. 64D). The
rCBF ratio in ROI-2 of the three groups was similar at 6 hrs, 1 and
3 days post MCAO. However, the rCBF ratio of both BMSC-treated and
PMSC-treated groups was increased at 7 days after MCAO as compared
to control (FIG. 64E). The rCBF ratio in ROI-3 was similar for the
three groups at 6 hrs, 1, and 3 days, but again the MSC groups had
a greater rCBF ratio at 7 days (FIG. 64F). In ROI-4, the rCBF ratio
slightly decreased in all groups at all time points, but not more
than 20 % (FIG. 64G).
[0578] [Example 78] Analysis of capillary in confocal images
[0579] To examine whether the administration of BMSCs and PMSCs
induces angiogenesis, three-dimensional analysis of capillary
vessels in the lesion was performed using Zeiss LSM5 PASCAL
software. FIG. 7A shows the three dimensional capillary image in
the normal rat brain. The capillary vascular volume in ROI-3 seven
days after MCAO was increased in both BMSC-treated (FIG. 65C) and
PMSC-treated groups (FIG. 65D) compared to the medium-treated group
(FIG. 65B). The capillary vascular volume was expressed as a ratio
by dividing that obtained from the ischemic hemisphere by that of
the contralateral control hemisphere. The ratio was significantly
higher in both the BMSC-treated (0.62.+-.0.05, n=5; p<0.05) and
the PMSC-treated (0.61.+-.0.05, n=5; p<0.05) groups as compared
to the medium-treated group (0.30.+-.0.02, n=5).
[0580] [Example 79] Functional analysis
[0581] To access behavioral performance in the lesioned and
transplanted animals, the treadmill stress test was used (FIG. 66).
Behavioral testing began 24 hours after lesion induction alone or
with cell transplantation. In the treadmill stress test control
animals (no lesion) reach a maximum treadmill velocity of about 70
m/min..sup.2 Twenty-four hours after MCAO without transplantation,
maximum velocity on the treadmill test was 10.0.+-.6.54 m/min
(n=5). Non-treated animals showed increased treadmill velocity with
slow improvement up to7 days (20.8.+-.10.9 m/min, n=5). In both
BMSCs and PMSCs transplantation groups, the improvement in velocity
was greater over the time course up to 7 days.
[0582] Industrial Applicability
[0583] The present inventors found that regenerative medicines with
the excellent features outlined below can be performed using
mesenchymal cells (mesenchymal stem cells), specifically bone
marrow cells, cord blood cells, or peripheral blood cells.
Specifically, regenerative treatments using bone marrow cells, cord
blood cells, or peripheral blood cells, using simple in vivo
administration (e.g., intravenous administration) of
patient-derived bone marrow cells by injection or drip infusion,
enable the regeneration of sites damaged by nervous system
injuries, and therapies for disorders . Such nervous system
injuries cannot in fact be treated by conventional techniques. The
efficacy of the present treatments on cerebral infarction has been
rigorously verified. Additionally, these treatments are considered
effective for all neurological diseases, such as injuries of the
nervous system due to cerebral infarction, intracerebral bleeding,
spinal cord injury, myocardial infarction, cerebral stroke
including subarachnoidal hemorrhage, central and peripheral
demyelination diseases, central and peripheral degenerative
diseases, brain tumor, higher-finction disorders including
dementia, mental disorders, epilepsia, traumatic neurological
diseases including head injuries, cerebral contusion, spinal cord
injuries, inflammatory diseases, and brain cell-damaging infective
diseases including Creutzfeldt-Jakob disease. The treatments can be
conducted in both specialized therapy facilities and in general
therapy sites (such as general hospitals, during ambulance
transportation, or at the site of incidents). These are
revolutionary treatments, since they enable therapies of disorders
that cannot be treated by conventional treatments, and further,
these treatments can be achieved using a simple procedure, such as
intravenous administration. In addition, since neurological
injuries cause severe disorders in patients, patients can benefit
tremendously from therapies for these disorders, which is of
tremendous social significance.
[0584] The medical action mechanisms of the regenerative medicines
of the present invention are as follows: Transplanted bone marrow
cells or mesenchymal stem cells migrate to and fixate in an
affected area (an in vivo injury site), recovering the functions of
the affected area by secreting appropriate substances, accelerating
inherent autotherapy, or differentiating into appropriate cells.
Accordingly, the regenerative medicines of the present invention
can exert effects on all types of diseases and events accompanied
by neurological injury.
Sequence CWU 1
1
10 1 40 DNA Artificial Sequence Description of Artificial Sequence
Synthetic primer 1 cggaattcca ccatgaccat ccttttcctt actatggtta 40 2
34 DNA Artificial Sequence Description of Artificial Sequence
Synthetic primer 2 ccagatctat cttccccttt taatggtcaa tgta 34 3 25
DNA Artificial Sequence Description of Artificial Sequence
Synthetic primer 3 tggaatcctg tggcatccat gaaac 25 4 25 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
primer 4 taaaacgcag ctcagtaaca gtccg 25 5 25 DNA Artificial
Sequence Description of Artificial Sequence Synthetic primer 5
cttagtctgg aggtggctac ataca 25 6 25 DNA Artificial Sequence
Description of Artificial Sequence Synthetic primer 6 gaggatagca
gaagaactag gcact 25 7 24 DNA Artificial Sequence Description of
Artificial Sequence Synthetic primer 7 ggtcacttca catgccatag tcaa
24 8 23 DNA Artificial Sequence Description of Artificial Sequence
Synthetic primer 8 ggctcagttg gtactttgcg taa 23 9 22 DNA Artificial
Sequence Description of Artificial Sequence Synthetic primer 9
attccgcgcc tctccctgtc tc 22 10 22 DNA Artificial Sequence
Description of Artificial Sequence Synthetic primer 10 gcttcatccg
cctcctgtct gt 22
* * * * *