U.S. patent application number 14/695843 was filed with the patent office on 2016-03-24 for pluripotent stem cell for treatment of cerebral infarction.
The applicant listed for this patent is CLIO, INC., TOHOKU UNIVERSITY. Invention is credited to Mari Dezawa, Teiji Tominaga, Masanori Yoshida.
Application Number | 20160082048 14/695843 |
Document ID | / |
Family ID | 54183439 |
Filed Date | 2016-03-24 |
United States Patent
Application |
20160082048 |
Kind Code |
A1 |
Yoshida; Masanori ; et
al. |
March 24, 2016 |
PLURIPOTENT STEM CELL FOR TREATMENT OF CEREBRAL INFARCTION
Abstract
An object of the present invention is to provide a novel medical
application to regenerative medicine that uses pluripotent stem
cells (Muse cells). The present invention provides a cell
preparation for treating cerebral infarction and sequelae
associated therewith that contains SSEA-3-positive pluripotent stem
cells isolated from mesenchymal tissue in the body or cultured
mesenchymal cells. The cell preparation of the present invention is
based on a brain tissue regeneration mechanism by which Muse cells
differentiate into nerve cells and the like in damaged brain tissue
by administering Muse cells into cerebral parenchyma.
Inventors: |
Yoshida; Masanori; (Akita
Shi, JP) ; Dezawa; Mari; (Sendai-shi, JP) ;
Tominaga; Teiji; (Sendai-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CLIO, INC.
TOHOKU UNIVERSITY |
Akita-shi
Sendai-shi |
|
JP
JP |
|
|
Family ID: |
54183439 |
Appl. No.: |
14/695843 |
Filed: |
April 24, 2015 |
Current U.S.
Class: |
435/325 |
Current CPC
Class: |
A61P 25/00 20180101;
A61P 9/10 20180101; A61P 43/00 20180101; A61K 35/545 20130101; A61P
9/00 20180101; A61P 25/28 20180101; A61K 35/28 20130101 |
International
Class: |
A61K 35/28 20060101
A61K035/28 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 26, 2014 |
JP |
2014-035725 |
Claims
1. A cell preparation for treating cerebral infarction, containing
pluripotent stem cells positive for SSEA-3 isolated from biological
mesenchymal tissue or cultured mesenchymal cells.
2. The cell preparation according to claim 1, for preventing and/or
treating sequelae from cerebral infarction.
3. The cell preparation according to claim 1, wherein the
pluripotent stem cells positive for SSEA-3 contain a concentrated
cell fraction as a result of stimulation by external stress.
4. The cell preparation according to claim 1, wherein the
pluripotent stem cells are CD105-positive.
5. The cell preparation according to claim 1, wherein the
pluripotent stem cells are CD117-negative and CD146-negative.
6. The cell preparation according to claim 1, wherein the
pluripotent stem cells are CD117-negative, CD146-negative,
NG2-negative, CD34-negative, vWF-negative and CD271-negative.
7. The cell preparation according to claim 1, wherein the
pluripotent stem cells are CD34-negative, CD117-negative,
CD146-negative, CD271-negative, NG2-negative, vWF-negative,
Sox10-negative, Snail-negative, Slug-negative, Tyrp1-negative and
Dct-negative.
8. The cell preparation according to claim 1, wherein the
pluripotent stem cells are pluripotent stem cells having all of the
properties indicated below: (i) low or absent telomerase activity;
(ii) ability to differentiate into any of the three germ layers;
(iii) absence of demonstration of neoplastic proliferation; and,
(iv) self-renewal ability.
9. The cell preparation described in claim 1, wherein the
pluripotent stem cells have the ability to differentiate into one
or more cells selected from the group consisting of nerve cells,
glial cells, vascular endothelial cells, and/or microglial cells.
Description
[0001] The present invention relates to a cell preparation used in
regenerative medicine. More specifically, the present invention
relates to a cell preparation containing pluripotent stem cells
that are effective for repairing and regenerating brain tissue that
has been damaged by cerebral infarction.
BACKGROUND OF THE INVENTION
[0002] Cerebral infarction refers to a brain dysfunction that
occurs due to ischemic necrosis localized in the brain, requires
emergency treatment, and is one of the three leading causes of
death after cancer and heart disease. In terms of its mechanism of
action, cerebral infarction is categorized as thrombotic, embolic
or hemodynamic cerebral infarction, or is classified into
categories such as atherothrombotic cerebral infarction,
cardiogenic embolism or lacunar infarction from the perspective of
clinical findings.
[0003] Ischemia occurs due to a local interruption of cerebral
blood flow caused by a cerebrovascular lesion such as
arteriosclerosis or cardiogenic thromboembolism, and nerve cell
death is induced at the ischemic core due to energy depletion. In
the area around the ischemic core, blood flow remains by means of
collateral circulation, and although nerve cells are not
functioning in terms of electrophysiology, they remain in a viable
state. Nerve cells in this area eventually end up undergoing
necrosis unless treatment is performed, and in pathological terms,
the infracted area progresses and clinically results in the
disorder of cerebral dysfunction. Accordingly, cerebral dysfunction
can be treated provided the function of nerve cells in the affected
area can be restored as quickly as possible. This reversible region
of incomplete ischemia is referred to as the penumbra. The purpose
of treating the acute stage of cerebral infarction is to restore
the function of nerve cells in this penumbra region, and the
outcome thereof is dependent upon the degree of ischemia and its
duration. Namely, outcome is determined by how quickly blood flow
can be resumed to the penumbra region. Nerve cells in this penumbra
region are believed to be able to survive for 3 to 6 hours
following an attack. In addition, the allowed amount of time during
which the function of nerve cells in a penumbra region can be
restored by treatment is referred to as treatment time (Stroke,
Vol. 21, p. 637-676 (1990)).
[0004] At present, thrombolytic therapy using recombinant human
plasminogen activator (rt-PA), which has been approved in the U.S.
for use in the treatment of acute stage cerebral infarction, was
developed for the purpose of restoring blood flow to a penumbra
region by lysing thrombi causing ischemia. In studies on rt-PA
therapy by intravenous injection targeted at cerebral infarction
patients within 3 hours after an attack, outcomes were
significantly more favorable after 3 months in an rt-PA dose group
in a double-blind placebo-controlled clinical trial conducted in
the U.S. rt-PA is thought to improve dysfunction caused by cerebral
infarction by enabling resumption of the supply of blood to an
ischemic region and inhibiting the progression of cerebral
infarction by lysing thrombi. This result indicated that early
resumption of cerebral blood flow by thrombolytic action improves
long-term prognosis (N. Eng. J. Med., Vol. 333, p. 1581-1587
(1995)). In addition, although stem cell therapy is expected to be
a new treatment method used in the treatment of cerebral infarction
in addition to using thrombolytic therapy as described above, it
has yet to demonstrate adequate therapeutic effects and is
currently not established as a treatment method (Sinden, J. D.
& Muir, K. W., Vol. 7, p. 426-434 (2012)).
[0005] It has been determined from research by M. Dezawa, one of
the inventors of the present invention, that
multilineage-differentiating stress enduring cells (Muse cells)
expressing surface antigen in the form of stage-specific embryonic
antigen-3 (SSEA-3), which are present in mesenchymal cell fractions
and can be obtained without going through an induction procedure,
are responsible for the pluripotency possessed by mesenchymal cell
fractions, and that they have the potential for application to
disease treatment aimed at tissue regeneration. In addition, Muse
cells were also determined to be able to be concentrated by
stimulating mesenchymal cell fractions with various types of stress
(WO2011/007900; Kuroda, Y., et al., Proc. Natl. Acad. Sci. USA,
Vol. 107, p. 8639-8643 (2010); Wakao, S., et al., Proc. Natl. Acad.
Sci. USA, Vol. 108, p. 9875-9880 (2011); Kuroda, Y., et al., Nat.
Protoco., Vol. 8, p. 1391-1415 (2013)). However, there have yet to
be any examples of the use of Muse cells for the treatment of
cerebral infarction, and the obtaining of anticipated therapeutic
effects has yet to be clearly determined.
SUMMARY
[0006] An object of the present invention is to provide a novel
medical application to regenerative medicine that uses pluripotent
stem cells (Muse cells). More specifically, an object of the
present invention is to provide a cell preparation for prevention
and/or treatment of cerebral infarction and sequelae occurring in
association therewith (including movement impairment, sensory
impairment and speech impairment) that contains Muse cells.
Means for Solving the Problems
[0007] The inventors of the present invention found that, by
injecting Muse cells into the cerebral parenchyma of a rat cerebral
infarction model induced by ischemia-reperfusion by inserting an
embolus into a cerebral blood vessel, the Muse cells survive over
the course of several months after taking to damaged brain tissue
and bring about a reduction in infarct size along with improvement
or restoration of brain function as a result of spontaneously
differentiating into brain cells, thereby leading to completion of
the present invention.
[0008] Namely, the present invention is as described below.
[0009] [1] A cell preparation for treating cerebral infarction,
containing pluripotent stem cells positive for SSEA-3 isolated from
biological mesenchymal tissue or cultured mesenchymal cells.
[0010] [2] The cell preparation according to [1] and [2] above, for
preventing and/or treating sequelae from cerebral infarction.
[0011] [3] The cell preparation according to [1] above, wherein the
pluripotent stem cells positive for SSEA-3 contain a concentrated
cell fraction as a result of stimulation by external stress.
[0012] [4] The cell preparation according to [1] to [3] above,
wherein the pluripotent stem cells are CD105-positive.
[0013] [5] The cell preparation according to [1] to [4] above,
wherein the pluripotent stem cells are CD117-negative and
CD146-negative.
[0014] [6] The cell preparation according to [1] to [5] above,
wherein the pluripotent stem cells are CD117-negative,
CD146-negative, NG2-negative, CD34-negative, vWF-negative and
CD271-negative.
[0015] [7] The cell preparation according to [1] to [6] above,
wherein the pluripotent stem cells are CD34-negative,
CD117-negative, CD146-negative, CD271-negative, NG2-negative,
vWF-negative, Sox10-negative, Snail-negative, Slug-negative,
Tyrp1-negative and Dct-negative.
[0016] [8] The cell preparation according to [1] to [7] above,
wherein the pluripotent stem cells are pluripotent stem cells
having all of the properties indicated below:
[0017] (i) low or absent telomerase activity;
[0018] (ii) ability to differentiate into any of the three germ
layers;
[0019] (iii) absence of demonstration of neoplastic proliferation;
and,
[0020] (iv) self-renewal ability.
[0021] [9] The cell preparation described in [1] to [8] above,
wherein the pluripotent stem cells have the ability to
differentiate into one or more cells selected from the group
consisting of nerve cells, glial cells, vascular endothelial cells,
and/or microglial cells.
[0022] The present invention is able to dramatically reduce infarct
size by a mechanism involving regeneration of brain tissue in which
Muse cells differentiate cells that compose brain tissue in damaged
brain tissue by being administered to the cerebral parenchyma of a
subject suffering from cerebral infarction.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 indicates the results of evaluating neurological
severity score (NSS) over the course of three months following
injection of Muse cells derived from human skin fibroblasts, human
skin fibroblasts from which Muse cells had been removed (namely,
non-Muse cells) or phosphate-buffered saline (PBS) into the
cerebral parenchyma of a rat cerebral infarction model. A decrease
in score value on the vertical axis corresponds to recovery of
brain function.
[0024] FIG. 2 indicates the results of a Rotarod performance test
of motor function in a rat cerebral infarction model injected with
Muse cells, non-Muse cells or phosphate-buffered saline (PBS).
Recovery of motor function was observed over time based on the
percentage of the average value of measured values (two) obtained
on two measurement days by using two measured values obtained prior
to transplant of Muse cells and the like as a baseline.
[0025] FIG. 3 indicates the results of measuring somatosensory
evoked potential (SEP) in a rat cerebral infarction model 85 days
after injecting Muse cells and the like.
[0026] FIG. 4 indicates fluorescent images indicating taking and
differentiation of Muse cells in brain tissue. Human-derived Muse
cells were labeled green with human mitochondria marker, while
nerve cells were labeled red using .beta.-tubulin III as a marker.
Muse cells injected into cerebral parenchyma were suggested to
accumulate in a region bordering the infarction and differentiate
into nerve cells after taking to that region. On the other hand,
taking and differentiation of non-Muse cells were not observed.
[0027] FIG. 5 indicates the results of observing human mitochondria
marker-positive cells under a fluorescence microscope and
respectively counting the number of cells contained in ten fields.
Although hardly any non-Muse cells took to a region bordering the
infarction, numerous Muse cells were present at that site.
DETAILED DESCRIPTION
[0028] The present invention relates to a cell preparation for
treating cerebral infarction that contains SSEA-3-positive
pluripotent stem cells (Muse cells). The following provides a
detailed explanation of the present invention.
1. Applicable Diseases
[0029] The present invention aims to treat cerebral infarction
using a cell preparation containing SSEA-3-positive pluripotent
stem cells (Muse cells). Here, "cerebral infarction" is a state
that ischemic area is locally generated in brain by obstruction or
perfusion pressure decrease of cerebral blood vessel whereby an
irreversible necrosis of neurons occurs. It is preferably an acute
brain infarction phase within 48 hours of the infarction onset,
more preferably within 24 hours, still more preferably within 6
hours and, most preferably, cerebral infraction within 3 hours of
the infraction onset. Here, the term "onset" is defined as the time
that which the patient was last seen in a normal state, or bedtime
for unwitnessed cerebral infraction occurring during the night. Due
to the cause of thrombus, cerebral infarction is classified into
cerebral thrombus and cerebral embolism and the present invention
is useful for the therapy of cerebral thrombus and cerebral
embolism. The term "therapy of cerebral infraction" means an effect
of preventing the progress of infarct foci in an acute phase of
cerebral infraction, an effect of improving the dysfunction or the
subjective symptom accompanied by cerebral infarction and/or an
effect of preventing the occurrence of psychiatric symptom and
convulsion onset during a chronic phase. It further includes
prevention of recurrence of the onset of cerebral infraction. In
addition, according to the observation by CT prior to the
administration, degree of cerebral infarction may be classified
depending upon infarct size, extent of infarct foci (penetrating
branch and cortical branch), side of infarct (left, right or both),
region of infarct (anterior cerebral artery region, middle cerebral
artery region, posterior cerebral artery region, watershed region,
brain stem, cerebellum and others) and degree of edema. The term
"suppression of the progress of cerebral infarction" means an
effect that expansion of infarct nidus with a lapse of time after
the onset of ischemic event is suppressed as compared with the
untreated case. The term "reducing effect of cerebral infarction"
means that the volume of infract foci generated by cerebral
infarction, which was measured prior to administration of the cell
preparation according to the present invention, is reduced at an
evaluation point after a certain period of time following the
administration of the cell preparation. Further, the cell
preparation according to the present invention can be used in
prevention and/or treatment of sequelae from cerebral infarction.
Here, "sequelae" includes speech and language disorder, disturbance
of perception such as numbness, disorder of movement in a limb,
headache, vomiting, visual loss, deglutition disorder, articulation
disorder, dementia and the like.
2. Cell Preparation
(1) Pluripotent Stem Cells (Muse Cells)
[0030] The existence of the pluripotent stem cells used in the cell
preparation of the present invention in the body was discovered by
M. Dezawa, one of the applicants of the present invention, and the
cells were named "multilineage-differentiating stress enduring
(Muse) cells". Muse cells can be obtained from bone marrow, adipose
tissue (Ogura, F., et al., Stem Cells Dev., Nov. 20, 2013 (Epub)
(published on Jan. 17, 2014), or skin tissue such as dermal
connective tissue, and are sporadically present in the connective
tissue of various organs. In addition, these cells have both the
properties of pluripotent stem cells and mesenchymal stem cells,
and are identified as being double-positive for each of the cell
surface markers of pluripotent stem cells, "stage-specific
embryonic antigen-3 (SSEA-3)" and of mesenchymal stem cells such as
"CD105". Thus, Muse cells or cell populations containing Muse cells
can be isolated from body tissue, for example, by using these
antigen markers as indicators. Details regarding methods used to
isolate and identify Muse cells as well as their characteristics
are disclosed in International Publication No. WO2011/007900. In
addition, as has been reported by Wakao, et al. (2011, previously
cited), in the case of using a cell culture obtained by culturing
mesenchymal cells present in bone marrow, skin and the like as the
parent population of Muse cells, all cells positive for SSEA-3 are
known to be positive for CD105. Thus, in the cell preparation of
the present invention, in the case of isolating Muse cells from
biological mesenchymal tissue or cultured mesenchymal cells, Muse
cells can be purified and used simply by using SSEA-3 as an antigen
marker. Furthermore, in the present description, pluripotent stem
cells (Muse cells) able to be used in a cell preparation for
treating cerebral infarction (including sequelae) that have been
isolated from biological mesenchymal tissue or cultured mesenchymal
cells by using SSEA-3 as an antigen marker, or a cell population
containing Muse cells, may simply be described as "SSEA-3-positive
cells". In addition, In the present specification, "non-Muse cells"
means cells included in biological mesenchymal tissue or cultured
mesenchymal cells, which are not "SSEA-3-positive cells."
[0031] Simply speaking, Muse cells or cell populations containing
Muse cells can be isolated from biological tissue (such as
mesenchymal tissue) using antibody to the cell surface marker
SSEA-3 alone or using antibody to SSEA-3 and CD105, respectively.
Here, "biological tissue" refers to the biological tissue of a
mammal. In the present invention, although an embryo in a
development stage prior to a fertilized egg or blastula stage is
not included in biological tissue, an embryo in a development stage
in or after the fetus or blastula stage, including the blastula, is
included. Examples of mammals include, but are not limited to,
primates such as humans or monkeys, rodents such as mice, rats,
rabbits or guinea pigs as well as cats, dogs, sheep, pigs, cows,
horses, donkeys, goats and ferrets. The Muse cells used in the cell
preparation of the present invention are clearly distinguished from
embryonic stem (ES) cells and embryonic germ (EG) cells in that
they are directly collectable from biological tissue and are
non-tumorigenic. In addition, "mesenchymal tissue" refers to tissue
such as bone, synovial membrane, fat, blood, bone marrow, skeletal
muscle, dermis, ligaments, tendons, tooth pulp, umbilical cord,
umbilical cord blood, as well as tissues present in various organs.
For example, Muse cells can be obtained from bone marrow, skin or
fat tissue. For example, Muse cells are preferably used that have
been isolated from mesenchymal tissue collected from the living
body. In addition, Muse cells may also be isolated from cultured
mesenchymal cells such as fibroblasts or bone mallow mesenchymal
cells using the aforementioned isolation means. Furthermore, Muse
cells used in the cell preparation of the present invention may be
autologous or allogenic relative to the recipient who receives the
cell transplant.
[0032] As has been described above, although Muse cells or cell
populations containing Muse cells can be isolated from biological
tissue by using their property of being SSEA-3-positive and
CD105-positive, human adult skin is known to contain various types
of stem cells and precursor cells. However, Muse cells are not the
same as these cells. Examples of such stem cells and precursor
cells include skin-derived precursor (SKP) cells, neural crest stem
cells (NCSC), melanoblasts (MB), perivascular cells (PC),
endothelial precursor (EP) cells and adipose-derived stem cells
(ADSC). Muse cells can be isolated from these cells by using
"non-expression" of a unique marker as an indicator of these cells.
More specifically, Muse cells can be isolated by using
non-expression of at least one of 11 markers, such as 1, 2, 3, 4,
5, 6, 7, 8, 9, 10 or 11 markers, selected from the group consisting
of CD34 (marker for EP and ADSC), CD117 (c-kit) (MB marker), CD146
(PC and ADSC marker), CD271 (NGFR) (NCSC marker), NG2 (PC marker),
vWF factor (von Willebrand factor) (EP marker), Sox10 (NCSC
marker), Snail (SKP marker), Slug (SKP marker), Tyrp1 (MB marker)
and Dct (MB marker). For example, although not limited thereto,
Muse cells can be isolated by using non-expression of CD117 and
CD146 as an indicator, can be isolated using non-expression of
CD117, CD146, NG2, CD34, vWF and CD271 as an indicator, and can be
isolated by using non-expression of the aforementioned 11 markers
as an indicator.
[0033] In addition, Muse cells having the aforementioned
characteristics used in the cell preparation of the present
invention may have at least one property selected from the group
consisting of:
[0034] (i) low or absent telomerase activity;
[0035] (ii) ability to differentiate into any of the three germ
layers;
[0036] (iii) absence of demonstration of neoplastic proliferation;
and,
[0037] (iv) self-renewal ability.
In one aspect of the present invention, the Muse cells used in the
cell preparation of the present invention have all of the
aforementioned properties. Here, with respect to the aforementioned
(i), "low or absent telomerase activity" refers to telomerase
activity being low or being unable to be detected in the case of
having detected telomerase activity using, for example, the Trapeze
XL Telomerase Detection Kit (Millipore Corp.). "Low" telomerase
activity refers to having telomerase activity roughly equal to that
of human fibroblasts, for example, or having telomerase activity
that is 1/5 or less and preferably 1/10 or less in comparison with
Hela cells. With respect to the aforementioned (ii), Muse cells
have the ability to differentiate into the three germ layers
(endoderm, mesoderm and ectoderm) in vitro and in vivo, and by
inducing to differentiate by culturing in vitro, for example, can
differentiate into skin, liver, nerve, muscle, bone or fat and the
like. In addition, Muse cells may also demonstrate the ability to
differentiate into the three germ layers in the case of
transplanting in vivo into testes, for example. Moreover, Muse
cells also have the ability to migrate, graft and differentiate
into a damaged organ (such as the heart, skin, spinal cord, liver
or muscle) by being transplanted into the body by intravenous
injection. With respect to the aforementioned (iii), although Muse
cells proliferate in a suspension culture, they have the property
of discontinuing proliferation for about 10.about.14 days. In
adherent culture, their doubling time is approximately 1.3
days/cell division which is similar to human fibroblasts, and keep
proliferating until cell reach nearly to Heyflick limit. Thus, in
the case of having been transplanted into testes, have the property
of not becoming malignant for at least six months. In addition,
with respect to the aforementioned (iv), Muse cells have
self-renewal (self-replication) ability. Here, "self-renewal"
refers to culturing cells contained in an embryoid body-like cell
mass obtained by suspension culturing single Muse cell and allowing
them to reform an embryoid body-like cell mass from a single cell
again as well as to demonstrate spontaneous differentiation of
embryoid body-like cell mass into triploblastic cell lineages on
gelatin coated culture. Self-renewal may be carried out for one
cycle or repeated for a plurality of cycles.
(2) Preparation and Use of Cell Preparation
[0038] The cell preparation of the present invention, although not
limited thereto, is obtained by suspending Muse cells or a cell
population containing Muse cells obtained in the aforementioned (1)
in physiological saline or a suitable buffer (such as
phosphate-buffered physiological saline). In this case, in the case
the number of Muse cells isolated from autologous or allogenic
tissue is low, cells may be cultured prior to cell transplant and
allowed them to proliferate until a prescribed cell concentration
is obtained. Furthermore, as has been previously reported
(International Publication No. WO 2011/007900), since Muse cells do
not undergo neoplastic transformation, there is little likelihood
of the cells becoming malignant even if cells recovered from
biological tissue are contained that have still not differentiated,
thereby making them safe. In addition, although there are no
particular limitations thereon, culturing of recovered Muse cells
can be carried out in an ordinary growth medium (such as minimum
essential medium-.alpha. (.alpha.-MEM) containing 10% bovine calf
serum). More specifically, a solution containing a prescribed
concentration of Muse cells can be prepared by selecting media,
additives (such as antibiotics and serum) and the like suitable for
the culturing and proliferation of Muse cells with reference to the
aforementioned International Publication No. WO2011/007900. In the
case of administering the cell preparation of the present invention
to a human, roughly several milliliters of bone marrow aspirate are
collected from human ilium, and after isolating Muse cells by using
an antigen marker for SSEA-3 as an indicator, the cells are allowed
to proliferate by culturing for an appropriate amount of time until
an effective therapeutic dose is reached, followed by preparing
autologous or allogenic Muse cells in the form of a cell
preparation. Alternatively, for instance, Muse cells are isolated
by using an antigen marker for SSEA-3 as an indicator, and then
after the cells are allowed to proliferate by culturing for an
appropriate amount of time until an effective therapeutic dose is
reached, autologous or allogenic Muse cells can be prepared as a
cell preparation.
[0039] In addition, when using the cell preparation of Muse cells,
dimethylsulfoxide (DMSO) or serum albumin for protecting the cells,
or antibiotics and the like for preventing contamination and growth
of bacteria, may also be contained in the cell preparation.
Moreover, other pharmaceutically allowable components (such as a
carrier, vehicle, disintegrating agent, buffer, emulsifier,
suspending agent, soothing agent, stabilizer, storage agent,
preservative or physiological saline), or cells or components other
than Muse cells contained in mesenchymal cells, may also be
contained in the cell preparation. A person with ordinary skill in
the art is able to add these factors and pharmaceutical agents to a
cell preparation at suitable concentrations. In this manner, Muse
cells can be used in the form of a pharmaceutical composition
containing various types of additives.
[0040] The number of Muse cells contained in the cell preparation
prepared in the manner described above can be suitably adjusted in
consideration of the gender, age and body weight of the subject,
disease state and state in which the cells are used so as to obtain
the desired effect in treatment of cerebral infarction and sequelae
(such as suppression of the progress of cerebral infarction,
reduction of cerebral infarction volume, restoration of motility
function, restoration of speech and language function, restoration
of perceptual function). In Examples 3 and 4 to be subsequently
described, a rat model of cerebral infarction was produced using an
embolus, and various types of effects of transplanting Muse cells
were examined. Extremely superior effects were obtained by
administering SSEA3-positive cells to Wistar rats weighing about
200 to 300 g at 3.times.10.sup.4 cells/animal. On the basis of this
result, superior effects can be expected to be obtained by
administering 1 to 1.5.times.10.sup.5 cells/kg per individual
mammal based on body weight. Here, examples of individuals include,
but are not limited to, rats and humans. In addition, the cell
preparation of the present invention may be administered a
plurality of times (such as 2 to 10 times) at a suitable interval
(such as twice per day, once per day, twice per week, once per
week, once every two weeks, once every one month, one every two
months, once every six months) until the desired therapeutic effect
is obtained. Thus, although dependent upon the status of the
subject, the therapeutically effective dose is preferably
administered, for example, 1 to 10 times at 1.times.10.sup.3 cells
to 2.times.10.sup.7 cells per individual. Although there are no
particular limitations thereon, examples of total individual doses
include 1.times.10.sup.3 cells to 2.times.10.sup.8 cells,
1.times.10.sup.4 cells to 1.times.10.sup.8 cells, 2.times.10.sup.4
cells to 5.times.10.sup.7 cells, 5.times.10.sup.4 cells to
2.times.10.sup.7 cells and 1.times.10.sup.5 cells to
1.times.10.sup.7 cells.
3. Preparation of Rat Cerebral Infarction Model
[0041] In the present description, a rat cerebral infraction model
can be constructed and used to examine the therapeutic effects of
the cell preparation of the present invention on cerebral
infarction (including sequelae). Although there are no particular
limitations of the rats of this model, typical examples thereof
include Wistar rats and Spraque-Dawley rats. A cerebral infarction
model can be created in order to promote symptoms resembling human
cerebral infarction by inserting an embolus from the carotid artery
of the rat, occluding the artery (such as the middle cerebral
artery) leading to the brain tissue where cerebral infarction is to
be induced for a prescribed amount of time with the embolus (to
induce an ischemic state), and then extracting the embolus.
Furthermore, the status of the cerebral infarction can be confirmed
with a brain tissue section (following TTC staining). In addition,
the cell preparation of the present invention has a heterologous
relationship with the rats administered with the preparation since
the Muse cells are of human origin. Normally, in experiments in
which heterologous cells are administered to model animals, an
immunosuppressant (such as cyclosporin) is administered either
prior or simultaneous to administration of the heterologous cells
in order to suppress an immune response in the body caused by the
heterologous cells.
4. Therapeutic Effects of Muse Cells
[0042] In embodiments of the present invention, the cell
preparation of the present invention is able to restore or return
to normal brain function in patients suffering from cerebral
infarction or patients suffering from sequelae thereof. When used
in the present description, restoration of brain function refers to
alleviation and inhibition of the progression of various functional
disorders (including sequelae) accompanying cerebral infarction,
and preferably refers to alleviation of functional disorders to a
degree that they do not present a problem during the course of
daily life. In addition, returning of brain function to normal
refers to returning functional disorders (including sequelae) to
the state prior to the onset of cerebral infarction. In addition,
although there are no particular limitations thereon, evaluation of
restoration of brain function is typically carried out by
electrophysiological studies, neurological severity scores (NSS),
imaging examinations and pathology studies. Here,
"electrophysiological studies" refer to studies performed to carry
out functional evaluations of various organs, including the brain,
by observing a potential (waveform of an electrical signal) in
response to an electrical stimulus with a prescribed apparatus in
order to evaluate the function of the central nervous system,
peripheral nervous system, muscle and the like. For example, in a
study of the central nervous system (spinal cord), this potential
is referred to as the somatosensory evoked potential (SEP), and the
study consists of an examination for measuring the potential
induced when a response induced by sensory stimulation of the limbs
passes through a sensory pathway and is transmitted to the cerebral
cortex. As a result, the degree of functional recovery of the
central nervous system of a patient can be confirmed objectively
following administration of the cell preparation of the present
invention to the patient. In addition, the neurological severity
score (NSS) is used to evaluate the degree of function of a damaged
portion of the brain by scoring each parameter. An NSS for use in
rats has been indicated by Chen, J. et al. (Stroke, Vol. 32, p.
1005-1111 (2001)).
[0043] Although the following provides a more detailed explanation
of the present invention through examples thereof, the present
invention is in no way limited by these examples.
EXAMPLES
Example 1
Preparation of Rat Cerebral Infarction Model
[0044] The "Regulations for Animal Experiments and Related
Activities at Tohoku University" were strictly observed for the
experimental protocol using rats in the present example, and
experimental animals were prepared in accordance with these
regulations under the supervision of the Tohoku University
Experimental Animal Center. More specifically, a rat cerebral
infarction model was prepared by inserting an embolus from the
carotid artery of Wistar rats (males, age 10 weeks) and occluding a
portion of the cerebral blood vessels (such as the middle carotid
artery (MCA)). Subsequently, the embolus was extracted, the vessel
was reperfused, and the rats were then used in the following
experiments as a cerebral infarction model. Furthermore, the status
of cerebral infarction was confirmed with brain tissue sections
(following TTC staining). In addition, an immunosuppressant (FK506)
was administered to the cerebral infarction rats prior to cell
transplant since the transplanted Muse cells are heterologous with
respect to rats.
Example 2
Preparation of Muse Cells
[0045] Preparation of human Muse cells derived from human
fibroblasts was carried out in accordance with the method described
in International Publication No. WO 2011/007900. More specifically,
adhesive mesenchymal cells were cultured from human bone marrow
fluid and after the cells proliferated, Muse cells or cell
populations containing Muse cells were isolated by FACS as
SSEA-3-positive cells. In addition, non-Muse cells consisted of a
cell group negative for SSEA-3 present among the aforementioned
mesenchymal cells, and were used as a control. Subsequently, the
cells were adjusted to a prescribed concentration using
phosphate-buffered physiological saline or culture liquid, and were
used to evaluate the effects of Muse cells on brain function in the
rat cerebral infarction model as indicated below. Furthermore, in
the case of using cells obtained by culturing mesenchymal cells
such as bone marrow-derived mesenchymal cells as a population of
Muse cells, all SSEA-3-positive cells are known to be
CD105-positive cells as reported by Wakao, et al. (2011, ibid).
Example 3
Evaluation of Brain Function by Transplanting Muse Cells
[0046] The cerebral infarction rats prepared in Example 1 were
divided into three groups, and Muse cells (1.times.10.sup.4 cells,
2 .mu.l PBS at 3 locations), non-Muse cells (1.times.10.sup.4
cells, 2 .mu.l PBS at three locations) or physiological saline (6
.mu.l) were injected directly into the cerebral parenchyma on day 2
following reperfusion. Subsequently, improvement of rat motor
function was evaluated over time and cell kinetics were analyzed
after a prescribed amount of time.
(1) Comprehensive Evaluation by Neurological Severity Score
(NSS)
[0047] Various types of brain function disorders (such as
paralysis, sensory disorders or vision disorders) were evaluated
using the neurological severity score (NSS) (Chen, J., Stroke, Vol.
32, p. 1005-1111 (2001)) in the rats transplanted with cells as
described above for a period of three months following transplant.
In this evaluation using NSS, points were assigned for changes in
motor function and as a result, a maximum score of 18 represents
serious neurological dysfunction, while a score of 0 represents a
normal neurological state. More specifically, evaluations were
carried out on the following parameters consisting of: standing up
with the tail (one point for each parameter (maximum of 3 points)),
state when lying on the floor (0 to 3 points), sensory test (1 or 2
points), beam balance test (0 to 6 points) and reflex absence/motor
impairment (maximum of 4 points). The results for the NSS scores of
each group of rats (n=10) are shown in FIG. 1. In the non-Muse cell
dose group and physiological saline dose group, scores decreased
for about the first ten days after which scores tended to be
maintained at 6 to 8 points. In contrast, in the Muse cell dose
group, scores on day 20 were significantly lower in comparison with
the other groups, and the scores tended to decrease further at the
time the experiment was completed (80 days and beyond), with
significant differences observed in comparison with the other
groups.
(2) Rotarod Performance Test
[0048] Restoration of cerebral dysfunction by transplantation of
Muse cells was investigated using an apparatus commonly known to be
used as an apparatus for measuring coordination of motor function
and sense of balance in experimental animals. Evaluation in this
test was carried out by measuring the amount of time until a rat
fell off a rotating stand twice each day at a frequency of once a
week (on days 0 to 84). The results are shown in FIG. 2. In the
non-Muse cell dose group and physiological saline dose group,
although roughly a maximum of 70% of motor function was observed to
have been restored from days 21 to 28, motor function was not
restored to 100% beyond that time. In contrast, in the Muse cell
dose group, although scores temporarily decreased to 70% after
initially recovering to 90% on day 28, motor function was observed
to be restored to nearly 100% starting on day 56. On the basis of
the comprehensive evaluation by NSS and the results of the Rotarod
performance test as described above, Muse cells were suggested to
remarkably improve brain function in cerebral infarction rats.
(3) Electrophysiological Studies
[0049] Somatosensory evoked potential (SEP) was measured in the rat
cerebral infarction model 85 days after injection of Muse cells and
the like (FIG. 3). The straight muscle of the thigh was stimulated
at 10 mA and 1 Hz 100 times (at 1 second intervals), and potential
was measured at a location 2.5 mm to the side and 2.5 mm posterior
to the bregma at a depth of 1 mm. Right brain-left leg (rt-lt)
indicates the latent time of stimuli traveling to the impaired
side, while left brain-left leg (lt-lt) indicates the latent time
of stimuli traveling to the same side of the brain, namely the
healthy side. A shorter latent time indicates rapid recovery. In
the group administered Muse cells, latent time was shorter than in
the PBS or non-Muse cell group for both right brain-left leg
(rt-lt) and left brain-left leg (lt-lt), and although statistically
significant differences were not observed, the measured values
suggested recovery of the neural network.
Example 4
Taking and Differentiation of Muse Cells in Brain Tissue
[0050] A study was conducted as to whether or not Muse cells take
to and differentiate into brain tissue in order to investigate the
behavior of Muse cells and non-Muse cells injected into cerebral
parenchyma. Brain tissue sections were prepared 85 days after
administering these cells and then observed under a fluorescence
microscope (FIG. 4). The cell nuclei of these sections were stained
with DAPI followed by double-staining with human mitochondria
marker and a nerve cell marker in the form of .beta.-tubulin III.
As a result, since fluorescence of the human mitochondria marker
(green) and fluorescence of the .beta.-tubulin III (red) indicating
nerve cells were observed in the same cell group in brain sections
of rats injected with Muse cells, Muse cells were suggested to take
to brain tissue and differentiate into nerve cells. On the other
hand, taking of non-Muse cells to brain tissue was not observed in
brain tissue sections in the case of having injected non-Muse
cells. In addition, in order to investigate the taking of Muse
cells and non-Muse cells to a region bordering the infarction,
human mitochondria marker-positive cells were observed under a
fluorescence microscope and the numbers of each cell contained in
10 fields were respectively counted (FIG. 5). Although hardly any
non-Muse cells took to the region bordering the infraction,
numerous Muse cells were present. On the basis of these results,
Muse cells were suggested to take to regions bordering on an
infarction and differentiate into nerve cells in comparison with
non-Muse cells.
INDUSTRIAL APPLICABILITY
[0051] The cell preparation of the present invention is able to
regenerate brain cells (such as nerve cells or glial cells) at the
site of a cerebral infarction by being administered into the
cerebral parenchyma of a cerebral infarction model, is able to
reduce infarct size and improve brain function, and can be applied
to the treatment of cerebral infarction and to the prevention
and/or treatment of sequelae following cerebral infarction.
[0052] All publications and patent documents cited in the present
description are incorporated throughout the description by
reference. Furthermore, although specific embodiments of the
present invention have been explained in the present description
for the purpose of exemplification, it can be easily understood by
a person with ordinary skill in the art that the present invention
may be modified in various ways without departing from the spirit
and scope thereof.
* * * * *