U.S. patent application number 14/238798 was filed with the patent office on 2015-02-12 for composition including stem cell-derived microvesicles for promoting neurogenesis.
This patent application is currently assigned to SAMSUNG LIFE PUBLIC WELFARE FOUNDATION. The applicant listed for this patent is Oh Young Bang, Yeon Hee Cho, Dong Hee Kim, Suk Jae Kim, Gyeong Joon Moon, Ji Hee Sung. Invention is credited to Oh Young Bang, Yeon Hee Cho, Dong Hee Kim, Suk Jae Kim, Gyeong Joon Moon, Ji Hee Sung.
Application Number | 20150045298 14/238798 |
Document ID | / |
Family ID | 47897528 |
Filed Date | 2015-02-12 |
United States Patent
Application |
20150045298 |
Kind Code |
A1 |
Bang; Oh Young ; et
al. |
February 12, 2015 |
COMPOSITION INCLUDING STEM CELL-DERIVED MICROVESICLES FOR PROMOTING
NEUROGENESIS
Abstract
The present invention relates to a composition including stem
cell-derived microvesicles as an active ingredient for promoting
neurogenesis. The stem cell-derived microvesicles according to the
present invention can promote neurogenesis and migration of nerves
and also promote angiogenesis in vascular endothelial cells, and
thus can be usefully used in treatment of neurological damage.
Inventors: |
Bang; Oh Young; (Gangnam-gu,
KR) ; Moon; Gyeong Joon; (Sujeong-gu, KR) ;
Cho; Yeon Hee; (Seongdong-gu, KR) ; Kim; Suk Jae;
(Songpa-gu, KR) ; Kim; Dong Hee; (Eunpyeong-gu,
KR) ; Sung; Ji Hee; (Gwanak-gu, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bang; Oh Young
Moon; Gyeong Joon
Cho; Yeon Hee
Kim; Suk Jae
Kim; Dong Hee
Sung; Ji Hee |
Gangnam-gu
Sujeong-gu
Seongdong-gu
Songpa-gu
Eunpyeong-gu
Gwanak-gu |
|
KR
KR
KR
KR
KR
KR |
|
|
Assignee: |
SAMSUNG LIFE PUBLIC WELFARE
FOUNDATION
Seoul
KR
|
Family ID: |
47897528 |
Appl. No.: |
14/238798 |
Filed: |
August 14, 2012 |
PCT Filed: |
August 14, 2012 |
PCT NO: |
PCT/KR2012/006478 |
371 Date: |
April 15, 2014 |
Current U.S.
Class: |
514/8.9 ;
514/17.7 |
Current CPC
Class: |
A61K 38/1793 20130101;
A61K 38/179 20130101; C12N 5/0623 20130101; C12N 2502/11 20130101;
A61P 9/10 20180101; A61P 25/28 20180101; C12N 5/069 20130101; C12N
2502/1358 20130101; A61P 9/00 20180101; A61P 25/08 20180101; A61P
25/00 20180101; A61K 35/51 20130101; A61K 35/12 20130101; A61P
25/16 20180101; A61K 35/28 20130101 |
Class at
Publication: |
514/8.9 ;
514/17.7 |
International
Class: |
A61K 35/28 20060101
A61K035/28; A61K 38/17 20060101 A61K038/17 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 26, 2011 |
KR |
10-2011-0081147 |
Aug 14, 2012 |
KR |
10-2012-0088778 |
Claims
1. A composition for promoting neurogenesis, comprising stem
cell-derived microvesicles as an active ingredient.
2. The composition for promoting neurogenesis of claim 1, wherein
the stem cell-derived microvesicles are microvesicles derived from
stem cells induced by ischemic stimuli.
3. The composition for promoting neurogenesis of claim 2, wherein
the ischemic stimuli are to treat stem cells with ischemic brain
extract.
4. The composition for promoting neurogenesis of claim 1, wherein
the stem cell-derived microvesicles are CD105-positive and annexin
V-negative microvesicles.
5. The composition for promoting neurogenesis of claim 1, wherein
the stem cells comprise at least one selected from the group
consisting of induced pluripotent stem cells (iPS), adult stem
cells, embryonic stem cells, mesenchymal stem cells, adipose stem
cells, hematopoietic stem cells, and cord blood stem cells.
6. The composition for promoting neurogenesis of claim 1, wherein
the cell-derived microvesicles promote neurogenesis and migration
of neuronal cells.
7. The composition for promoting neurogenesis of claim 1, wherein
the cell-derived microvesicles promote angiogenesis of vascular
endothelial cells.
8. A method for promoting neurogenesis, comprising the step of
applying stem cell-derived microvesicles to neural stem cells.
9. The method for promoting neurogenesis of claim 8, wherein the
stem cell-derived microvesicles are obtained by applying ischemic
stimuli to stem cells.
10. The method for promoting neurogenesis of claim 9, wherein the
ischemic stimuli are to treat stem cells with ischemic brain
extract.
11. The method for promoting neurogenesis of claim 8, wherein the
stem cells comprise at least one selected from the group consisting
of induced. pluripotent stem cells (iPS), adult stem cells,
embryonic stem cells, mesenchymal stem cells, adipose stem cells,
hematopoietic stem cells, and cord blood stem cells.
12. A pharmaceutical composition for prevention or treatment of
degenerative neurological diseases, comprising stem cell-derived
microvesicles as an active ingredient.
13. The pharmaceutical composition for prevention or treatment of
degenerative neurological diseases of claim 12, wherein the
degenerative neurological disease comprises at least one selected
from the group consisting of ischemic stroke, cerebral infarction,
neurotrauma, Parkinson's disease, Lou Gehrig' disease, and
epilepsy.
14. A method for treating neurological damage, comprising the step
of treating a subject other than human, suffering from neurological
damage, with stem cell-derived microvesicles.
15. The method for treating neurological damage of claim 14,
wherein the neurological damage is neurological damage caused by a
physical damage or degenerative neurological disease.
16. The method for treating neurological damage of claim 15,
wherein the physical damage comprises at least one selected from
the group: consisting of brain trauma, spinal cord injury, and
neurotrauma.
17. The method for treating neurological damage of claim 15,
wherein the degenerative neurological disease comprises at least
one selected from the group consisting of ischemic stroke, cerebral
infarction, neurotrauma, Parkinson's disease, Lou Gehrig' disease,
and epilepsy.
18. The method for treating neurological damage of claim 14,
wherein the subject other than human comprises at least one
selected from the group consisting of a rat, a red fox, a skunk, a
raccoon, a badger, a dog, a wolf, a mongoose, a coyote, a weasel,
and a cat.
19. The method for treating neurological damage of claim 14,
wherein the cell-derived microvesicles promote neurogenesis and
migration of neuronal cells.
Description
TECHNICAL FIELD
[0001] The present invention relates to a composition comprising
stem cell-derived microvesicles for promoting neurogenesis and a
method for promoting neurogenesis.
BACKGROUND ART
[0002] Recently, various preclinical and clinical studies on stem
cell therapies have been conducted for various diseases such as
cerebral infarction, traumatic neuronal injury, musculoskeletal
disease, etc. However, the current technology has reached up to the
level of simple extraction, culture and proliferation of stem cells
and injection of the stem cells. Moreover, it is known, as a result
of clinical studies up to now, that stem cell therapies have not
yet shown a significant effect. Extensive research on a variety of
genetically modified stem cells to promote the effect has continued
to progress, but cell therapies using genes cannot be applied to
the human body due to ethical issues.
[0003] Moreover, there are several problems in clinical application
of the use of stem cells. Firstly, in the case of cell therapy
products, there is a risk of tumor formation after stem cells are
transplanted into tissue. Secondly, stem cells may cause arterial
occlusion due to a relatively large size, resulting in cerebral
infarction. Thirdly, stem cells can migrate to the brain during the
acute phase when the brain-blood barrier is open but have
limitations in crossing the brain-blood barrier due to a large size
during the chronic stage. Lastly, inducing stem cells to
specialized cells having desired properties for cell therapy
products has limitation.
[0004] Contrarily, cell therapy using microvesicles has recently
attracted attention as a method that is differentiated from the
cell therapy using stem cells. Typically, microvesicles are small
vesicles of 0.1to 1 .mu.m diameter and refer to cell membrane
microparticles circulating in the blood, such as endothelial cells,
platelets, etc. It is known that stem cell-derived microvesicles
contain proteins, receptors as well as nuclear components and thus
have a role in cell-to-cell communication. Moreover, the
microvesicles contain a relatively small amount of animal serum
compared to stem cells, and the risk of zoonosis can also be
eliminated. In view of these characteristics of microvesicles, the
cell therapy using microvesicles is expected to be a new paradigm
that can overcome the limitations of existing stem cell
therapies.
[0005] Therefore, extensive research aimed at using these stem
cell-derived microvesicles instead of stem cells has continued to
progress. For example, International Patent Publication No. WO
2010/070141 discloses a method for producing stem cell-derived
microvesicles and therapeutic effects of the produced microvesicles
on immune disease, allergic response, inflammatory disease, etc.,
and Korean Patent Publication No. 2010-122027 discloses a particle
secreted by a mesenchymal stem cell and comprising at least one
biological property of a mesenchymal stem cell and a technology
using the particle as a therapeutic agent for cardioprotection.
[0006] However, there is not much research on stem cell-derived
microvesicles, and it cannot be said that all stem cells that can
be used for treatment of various diseases can be replaced by
microvesicles. In particular, the correlation between microvesicles
and neurogenesis has not been reported.
[0007] Therefore, if the generation of neuronal cells can be
promoted using stem cell-derived microvesicles, it is possible to
use them in the treatment of diseases related to neurological
damage, and thus there is an urgent need to study the correlation
between microvesicles and neuronal cells.
DISCLOSURE
Technical Problem
[0008] The present inventors have studied the correlation between
microvesicles and neuronal cells and found that stem cell-derived
microvesicles have excellent effects of promoting neurogenesis and
migration of neuronal cells and promoting angiogenesis in vascular
endothelial cells, thus completing the present invention.
Technical Solution
[0009] To achieve the above objects, the present invention provides
a composition for promoting neurogenesis, comprising stem
cell-derived microvesicles as an active ingredient.
[0010] Moreover, the present invention provides a method for
promoting neurogenesis, comprising the step of applying stem
cell-derived microvesicles to neural stem cells.
[0011] Furthermore, the present invention provides a pharmaceutical
composition for prevention or treatment of degenerative
neurological diseases, comprising stem cell-derived microvesicles
as an active ingredient.
[0012] In addition, the present invention provides a method for
treating neurological damage, comprising the step of treating a
subject other than human, suffering from neurological damage, with
stem cell-derived microvesicles.
Advantageous Effects
[0013] The stem cell-derived microvesicles according to the present
invention have excellent effects of promoting neurogenesis and
migration of neuronal cells and promoting angiogenesis of vascular
endothelial cells, thus treating neurological damage.
DESCRIPTION OF DRAWINGS
[0014] FIG. 1 shows blood levels of CD105-positive/annexin
V-negative and CXCR4-positive/annexin V-negative microvesicles in a
patient with a small infarct (A) and in a patient with a large
infarct (B) and the correlation between CD105-positive and
CD90-positive (C).
[0015] FIG. 2 shows the correlation between CD105-positive/annexin
V-negative microvesicles (A), CD105-positive/CXCR4-positive/annexin
V-negative microvesicles (B), and SDF-1.alpha. (C) according to the
infarct size (DWI), NIHSS, and the time since the onset of cerebral
infarction.
[0016] FIG. 3 shows the numbers of stem cell-derived microvesicles
in bone marrow-derived mesenchymal stem cells treated with Kn DEME
(knockout DEME), STS 100 nm (staurosporin A), 20% ischemic brain
extract (20% BE (whole)), and 20% ischemic serum (20% serum),
determined by a flow cytometer using CD105 and annexin V.
[0017] FIG. 4 shows neuronal cell deaths atter treatment of
neuronal cells with stem cell-derived microvesicles (1, 3, 10, 30
.mu.g/ml) obtained by ischemic stimuli or with 30 .mu.m NMDA as a
control, determined by LDH analysis.
[0018] FIG. 5 shows neurogenesis capabilities after treatment of
neuronal stem cells with microvesicles obtained without ischemic
stimuli (A) or with stem cell-derived microvesicles obtained by
ischemic stimuli (B), determined by an optical microscope and a
confocal microscope.
[0019] FIG. 6 shows angiogenesis capabilities of vascular
endothelial cells after treatment with microvesicles (1, 3, 10, 30
.mu.m/ml) obtained by ischemic stimuli, determined by a
fluorescence microscope.
[0020] FIG. 7 shows migration distances of neural progenitor cells
after injection of microvesicles obtained by ischemic stimuli into
the ventricle of rats with ischemic stroke, determined by
fluorescence staining (A) and shows the numerical results thereof
(B) (Contra: contralateral, ipsi: ipsilateral).
[0021] FIG. 8 shows angiogenesis of vascular endothelial cells
after injection of stem cell-derived microvesicles obtained by
ischemic stimuli into the ventricle of rats with ischemic stroke,
determined by fluorescence staining (A) and shows the numerical
results thereof (B) (Contra: contralateral, ipsi: ipsilateral,
sham: normal control group).
MODE FOR INVENTION
[0022] The present invention provides a composition for promoting
neurogenesis, comprising stem cell-derived microvesicles as an
active ingredient.
[0023] Moreover, the present invention provides a method for
promoting neurogenesis, comprising the step of applying stem
cell-derived microvesicles to neural stem cells.
[0024] As used herein, the term "stem cell-derived microvesicles"
refers to small vesicles derived from stem cells, containing
receptors and proteins, and having a diameter of 0.1 to 1
.mu.m.
[0025] The stem cell-derived microvesicles may be microvesicles
derived from stem cells induced by ischemic stimuli, and the
ischemic stimuli may preferably be ischemic preconditioning
stimuli. The ischemic preconditioning stimuli may be performed by
exposing stem cells to an environment that causes ischemia or
treating stem cells with ischemic brain extract.
[0026] The ischemic brain extract may be an extract obtained from
the brain of subjects that have ischemic symptoms. The one example
of ischemic symptoms may be ischemic stroke.
[0027] The stem cell-derived microvesicles may include, but not
particularly limited to, CD105 positive microvesicles, annexin
V-negative microvesicles, etc. which are specific to mesenchymal
stem cells
[0028] Here, the stem cells that are the source of the
microvesicles may be, but not limited to, any one of induced
pluripotent stem cells (iPS), adult stem cells, embryonic stem
cells, mesenchymal stem cells, adipose stem cells, hematopoietic
stem cells, and cord blood stem cells, preferably mesenchymal stem
cells.
[0029] The stem cell-derived microvesicles according to the present
invention can promote neurogenesis and migration of neuronal cells
and also promote angiogenesis in vascular endothelial cells.
[0030] Moreover, the present invention provides a pharmaceutical
composition for prevention or treatment of degenerative:
neurological diseases, comprising stem cell-derived microvesicles
as an active ingredient.
[0031] The stem cell-derived microvesicles according to the present
invention can promote neurogenesis and migration of neuronal cells
and promote angiogenesis in vascular endothelial cells, thus having
excellent effect of treating neurological damage. Therefore, the
stem cell-derived microvesicles according to the present invention
can be effectively used in prevention or treatment of degenerative
neurological diseases.
[0032] As used herein, the term "degenerative neurological
diseases" is intended to include, but not limited to, a variety of
diseases caused by neurological damage without limitation, and an
example of the degenerative neurological diseases may be any one of
ischemic stroke, cerebral infarction, neurotrauma, Parkinson's
disease, Lou Gehrig' disease, and epilepsy.
[0033] The pharmaceutical composition for prevention or treatment
of degenerative neurological diseases, comprising stem cell-derived
microvesicles as an active ingredient according to the present
invention may preferably contain other ingredients which may
provide a synergy effect to the main effect within a range of not
impairing the main effect of the present invention, in addition to
the stem cell-derived microvesicles.
[0034] Moreover, the pharmaceutical composition of the present
invention may further comprise pharmaceutically acceptable carrier,
excipients, and diluents for administration, in addition to the
above-described active ingredient. Examples of carriers,
excipients, and diluents may include lactose, dextrose, sucrose,
sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia
gum, alginate, gelatin, calcium phosphate, calcium silicate,
cellulose, methylcellulose, microcrystalline cellulose,
polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl
hydroxybenzoate, talc, magnesium stearate, and mineral oil.
[0035] The pharmaceutical composition of the present invention may
be formulated into various formulations for parenteral or oral
administration. A representative example of the parenteral
formulation may preferably be an isotonic aqueous solution or as an
injectable formulation. The injectable formulation may be prepared
by a method known in the art using a suitable dispersant or wetting
agent and a suspending agent by any method known in the art. For
example, the respective ingredients may be dissolved in a saline or
buffer solution to be formulated into an injectable
formulation.
[0036] Solid formulations for oral administration may include
tablets, pills, powders, granules, capsules, etc. The solid
formulations may be prepared by mixing the active ingredient with
at least one excipient such as starch, calcium carbonate, sucrose,
lactose, gelatin, etc. Moreover, lubricants such as magnesium
stearate, talc, etc. may be used in addition to simple
excipients.
[0037] Liquid formulations for oral administration may include
suspensions, internal solutions, emulsions, syrups, etc. The liquid
formulations may include, and various excipients such as
humectants, sweeteners, aromatics, preservatives, etc. may be
included in addition to generally-used simple diluents such as
water and liquid paraffin.
[0038] Formulations for parenteral administration may include
sterile solutions, non-aqueous solvents, suspensions, emulsions,
freeze-dried formulations, and suppositories. Propylene glycol,
polyethylene glycol, vegetable it such as olive oil, and injectable
ester such as ethyl oleate, etc. may be used for non-aqueous
solvents and suspensions. Witepsol, macrogol, Tween 61, cacao oil,
laurin oil, glycerogelatin, etc. may be used for suppository
bases.
[0039] The effective dosage of the pharmaceutical composition of
the present invention may vary depending on the age, gender, and
weight of a patient. However, the pharmaceutical composition of the
present invention may be administered in a dose of 100 .mu.g/kg to
1 mg/kg, preferably 200 .mu.g/kg to 500 .mu.g/kg.
[0040] For the prevention or treatment of degenerative neurological
diseases, the composition of the present invention may be used
alone or in combination with surgical operation, chemical therapy,
radiotherapy, hormonal therapy, chemical therapy, drug therapy, and
methods using biological response modifiers.
[0041] Moreover, the present invention provides a method for
treating neurological damage, comprising the step of treating a
subject other than human, suffering from neurological damage, with
stem cell-derived microvesicles.
[0042] The treatment with stem cell-derived microvesicles promotes
neurogenesis and migration of neuronal cells and also promotes
angiogenesis in vascular endothelial cells, thus effectively
treating neurological damage.
[0043] As used here, the term "neurological damage" refers to
damage to the nerve caused by physical factors or degenerative
neurological diseases. The neurological damage by physical factors
may include, but not limited to, brain trauma, spinal cord injury,
cerebral infarction, cerebral hemorrhage, neurotrauma, etc., and
the neurological damage by degenerative neurological disease may
include, but not limited to, ischemic stroke, cerebral infarction,
neurotrauma, Parkinson's disease, Lou Gehrig' disease, and
epilepsy.
[0044] The subject other than human may preferably include, but not
limited to, a rat, a red fox, a skunk, a raccoon, a badger, a dog,
a wolf, a mongoose, a coyote, a weasel, and a cat.
MODE FOR INVENTION
[0045] In the following, the present invention will be described in
detail with reference Preparation Examples and Examples. However,
the following Preparation Examples and
[0046] Examples are provided only for illustration of the present
invention, and the present invention is not limited by the
following Preparation Examples and Examples.
EXAMPLE 1
Comparison of the levels of Stem Cell-Derived Microvesicles in
Serum
[0047] Citrated sera were collected from stroke patients and
centrifuged at 19,800 g and 10.degree. C. for 10 minutes to obtain
stem cell-derived microvesicles. The obtained stem cell-derived
microvesicles were suspended in 20 .mu.l PBS, and the blood levels
of stem cell-derived microvesicles (CD105-positive/annexin
V-negative) and microvesicles (CXCR4-positive/annexin V-negative)
expressing the SDF-1 receptor, CXCR4, were compared in patients
with a small infarct (DWI volume: 10 cc or less) and patients with
a large infarct (DWI volume: 10 cc or more), and it was determined
whether CD105-positive cells were CD90-positive or double-positive
cells. The results of the above experiment are shown in FIG. 1.
[0048] As shown in FIG. 1, it was found that the amount of
CD105-positive cells and CXCR4-positive cells in patients with a
large infarct (B) after the onset of cerebral infarction was
greater than that in patients with a small infarct (A). It can be
seen from this result that the number of stem cell-derived
microvesicles and microvesicles capable of migrating by SDF-1
chemotaxis increases in patients with a large infarct. Moreover, it
was found that most CD105-positive cells were CD90 double-positive
cells, indicating that CD105-positive microvesicles were derived
from stem cells.
[0049] Meanwhile, based on clinical records of stroke patients, the
correlation between stem cell-derived microvesicles
(CD105-positive/annexin V-negative), microvesicles expressing the
SDF-1 receptor, CXCR4 (CXCR4-positive/annexin V-negative), and
SDF-1.alpha. according to the diffusion weighted image (DWI)
showing the infarct size, the National Institutes of Health Stroke
Scale (NIHSS), and the time since the onset of cerebral infarction
was analyzed. The results of the above experiment are shown in FIG.
2.
[0050] As shown in FIG. 2, the number of CD105-positive/annexin
V-negative stem cell-derived microvesicles showed a tendency to
increase as the infarct size was larger and the NIHSS score was
higher (A), and the number of CD105-positive/CXCR4-positive/annexin
V-negative microvesicles showed a tendency to increase as the NIHSS
score was higher and to decrease as the time since the onset of
cerebral infarction increased (B). It can be seen from these
results that the blood level of stem cells with high motility was
increased by microvesicles as the severity of cerebral infarction
was higher, indicating that the existence of potential stem cells
may decrease as the time since the onset of cerebral infarction
increases. Meanwhile:, the blood concentration of SDF-1.alpha.
decreased as the infarct size was larger and increased as the time
since the onset of cerebral infarction increased (C).
EXAMPLE 2
Isolation of Microvesicles from Bone Marrow-Derived Stem Cells
2.1 Collection and Culture of Mesenchymal Stem Cells
[0051] Bone marrows were collected from the femur of rats to obtain
bone marrow-derived mesenchymal stem cells by culture. The obtained
mesenchymal stem cells were cultured at a density of
1.times.10.sup.5 cell/ml in T75 flask at 5% CO.sub.2 and 37.degree.
C. and subcultured at 80% confluence. The subculture was performed
in a manner that the medium was removed, the stem cells were gently
washed with PBS more than once, cultured in a 37.degree. C.
incubator for about 1 minute after addition of Tryple (Invitrogen),
and placed in a medium containing 10% FBS fetal bovine serum (FBS)
to neutralize the reaction, and then the cells were collected and
centrifuged at 1,300 g for 4 minutes. The cell precipitates were
resuspended in a medium containing FBS, counted, and cultured. The
fourth to sixth passage cells were used in the experiment.
Low-glucose DMEM containing 10% PBS and 1% penicillin/streptomycin
was used as a medium.
2.2 Preparation of Simulatives for Ischemic Preconditioning
Stimuli
[0052] Hemispheric tissues of white rats with induced middle
cerebral artery occlusion were fragmented in 150 mg/ml DMEM medium
and then centrifuged at 10,000 g for 10 minutes, and the
supernatant was collected to obtain ischemic brain extracts. The
obtained ischemic brain extracts were plated in equal amounts and
kept at -70.degree. C. For application of ischemic stimuli to stem
cells, the stem cells were suspended in DMEM medium to prepare 20%
ischemic brain extract, thus preparing stimulatives for ischemic
preconditioning stimuli containing 20% ischemic brain extract or
20% ischemic serum.
2.3 Isolation and Analysis of Stem Cell-Derived Microvesicles
[0053] The bone marrow derived mesenchymal stem cells prepared in
Example 2.1 were cultured in a 60 mm culture dish in DMEM medium
containing 10% FBS. The stimulatives for ischemic preconditioning
stimuli (20% ischemic brain extract or 20% ischemic serum) prepared
in Example 2.2 were added, and then the supernatant was collected
from the culture medium. The supernatant was centrifuged at low
speed (2,500 g, 10.degree. C., 10 minutes) to remove impurities
from the supernatant and centrifuged again at high speed (14,500 g,
10.degree. C., 45 minutes), thus obtaining microvesicles derived
from stem cells. The number of the obtained microvesicle was
calculated by the following formula, and the expression degree of
CD105 and annexin V in each microvesicle was analyzed using a flow
cytometer.
The number of microvesicles (n/L)=(the total amount of microvesicle
suspension/the amount of microvesicles used in the antibody
response).times.(the total amount of microvesicle diluent for flow
cytometry/the amount of microvesicle diluent used for flow
cytometry).times.(10.sup.6/the total amount of serum)
[0054] The expression degree of CD105 and annexin V was used as a
marker to determine whether the microvesicles were derived from
mesenchymal stem cells, and microvesicles obtained from bone
marrow-derived mesenchymal stem cells treated with knockout DEME
and staurosporin A were used as controls. The results of the above
experiment are shown in FIG. 3.
[0055] As shown in FIG. 3, it can be seen that the amount of
microvesicles obtained from bone marrow-derived mesenchymal stem
cells did not increase in the control groups treated with Kn DEME
(knockout DEME), STS 100 nm (staurosporin A), and 20% ischemic
serum (20% serum), but the amount of microvesicles significantly
increased in the group treated with 20% ischemic brain extract (20%
BE (whole)). Therefore, it was found that it was desirable to use
the ischemic brain extract in order to increase the secretion of
microvesicles.
2.4 Determination of Safety of Stem Cell-Derived Microvesicles
[0056] It was determined whether cell death was induced when
neuronal cells under culture were treated with microvesicles
obtained by ischemic preconditioning. The neuronal cell death was
determined by LDH analysis 24 hours after treating neuronal cell
culture with microvesicles (1, 3, 10, 30 .mu.g/ml) obtained by
ischemic preconditioning or with 30 .mu.m NMDA positive control).
The results of the above experiment are shown in FIG. 4.
[0057] As shown in FIG. 4, it was found that microvesicles obtained
by ischemic preconditioning did not cause neuronal cell death, but
more than 70% neuronal cell death occurred in the group treated
with NMDA. Therefore, it can be seen that the microvesicles
obtained by ischemic preconditioning do not induce the death of
neural stem cells and this is considered as a safe material.
EXAMPLE 3
Stimulation of Neural Stem Cells using Stem Cell-Derived
Microvesicles
3.1 Isolation and Culture of Neural Stem Cells
[0058] Brain tissues collected from 14-day-old embryonic rats were
gently fragmented using a narrow-bore glass pipette or dissociated
to single cell level using a cell dissociation solution such as
Accutase and centrifuged (500 g, 5 minutes), thus obtaining
precipitated cells. The obtained cells were cultured in DMEM/F12
medium containing 1XN2 supplement, 25 ng/ml bFGF, and 25 ng/ml EGF
to induce neural stem cells. The induced neural stem cells were
subcultured such that the diameter of the neuronal cell is
maintained within 100 rim.
3.2 Increase in Neurogenesis Capability of Neural Stem Cells by
Stem Cell-Derived Microvesicles
[0059] The neural stem cells induced in Example 3.1 were treated
with the stem cell-derived microvesicles obtained in Example 2.3 by
ischemic preconditioning, and the neurogenesis capabilities of the
treated neural stem cells were compared. Microvesicles obtained
from mesenchymal stem cells without ischemic stimuli were used as a
control. Each experimental group was observed under an optical
microscope and a confocal microscope. Nestin as a marker for neural
stem cells and doublecortin (DCX) as a marker for neural progenitor
cells were used, and their differentiation into astroglial cells
was determined. The results of the above experiment are shown in
FIG. 5.
[0060] As shown in FIG. 5, it was found that the neurogenesis
capability did not significantly increase in neural stem cells
treated with microvesicles obtained without ischemic stimuli (A),
but the expression of nestin and DCX markers significantly
increased in neural stem cells treated with microvesicles obtained
by applying ischemic stimuli to bone marrow stem cells (B),
representing the increase in neurogenesis capability.
3.3 Increase in Angiogenesis Capability of Vascular Endothelial
Cells by Stem Cell-Derived Microvesicles
[0061] Vascular endothelial cells were treated with the
microvesicles (1, 3, 10, 30 .mu.g/ml) obtained in Example 2.2 by
ischemic stimuli, and the angiogenesis capabilities of vascular
endothelial cells were compared. Microvesicles obtained from
mesenchymal stem cells without ischemic stimuli were used as a
control. Living cells were fluorescence-stained with Calcein AM to
be used as a marker to measure the angiogenesis capability and
observed under a fluorescence microscope. The results of the above
experiment are shown in FIG. 6.
[0062] As shown in FIG. 6, it was found that the increase in
fluorescence was not observed in the control group (ctrl), but the
fluorescence increased in a concentration-dependent manner by
angiogenesis in vascular endothelial cells after treatment with
stem cell-derived microvesicles. Therefore, it can be seen that the
stem cell-derived microvesicles can promote angiogenesis in
vascular endothelial cells.
3.4 Stimulation of Neural Stem Cells by Stem Cell-Derived
Microvesicles in Ischemic Stroke Animals (In Vivo)
[0063] The microvesicles obtained in Example 2.3 by ischemic
stimuli were injected into the ventricle of rats with ischemic
stroke, and the migration distances of neural progenitor cells were
compared. PBS containing no microvesicles was injected into the
ventricle and used as a control, and the microvesicles were
fluorescence-stained with antibodies against the DCX marker for
neural progenitor cells.
[0064] The results are shown in FIG. 7.
[0065] As shown in FIG. 7, it was found that the injection of stem
cell-derived microvesicles into the ventricle of rats with ischemic
stroke significantly increased the migration distance of neural
progenitor cells in both the ipsilateral (ipsi) and contralateral
(contra) of the ventricle, compared to the control group (Veh)
treated PBS. Therefore, it can be seen that the stem cell-derived
microvesicles can stimulate neural progenitor cells to promote
their migration.
3.5 Promotion of Neurogenesis by Stem Cell-Derived Microvesicles in
Ischemic Stroke Animals (In Viva)
[0066] The microvesicles obtained in Example 2.3 by ischemic
stimuli were injected into the ventricle of rats with ischemic
stroke, and the degrees of angiogenesis of vascular endothelial
cells were compared. PBS containing no microvesicles was injected
into the ventricle and used as a control, and the microvesicles
were fluorescence-stained with antibodies against Von Willebrand
factor (vWF), a marker for neural progenitor cells. The results of
the above experiment are shown in FIG. 8.
[0067] As shown in FIG. 8, it was found that the neurogenesis
increased in both the ipsilateral (ipsi) and contralateral (contra)
of the ventricle and, in particular, significantly increased in the
contralateral of the ventricle in the experimental group treated
with stem cell-derived microvesicles, compared with the normal
control and the PBS-treated control group (sham). Therefore, it can
be seen that the stem cell-derived microvesicles can promote the
migration of neural progenitor cells and also promote angiogenesis
in vascular endothelial cells.
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