U.S. patent application number 14/018849 was filed with the patent office on 2014-03-20 for methods to treat nervous system conditions.
The applicant listed for this patent is Yansheng Du, Brian H. Johnstone, Keith Leonard March. Invention is credited to Yansheng Du, Brian H. Johnstone, Keith Leonard March.
Application Number | 20140079802 14/018849 |
Document ID | / |
Family ID | 39107733 |
Filed Date | 2014-03-20 |
United States Patent
Application |
20140079802 |
Kind Code |
A1 |
March; Keith Leonard ; et
al. |
March 20, 2014 |
METHODS TO TREAT NERVOUS SYSTEM CONDITIONS
Abstract
Methods to treat nervous system conditions. In at least one
embodiment of a method of treating a mammalian patient having a
neuronal injury or insult of the present disclosure, the method
comprises the step of administering a therapeutically-effective
dose of a stem cell conditioned medium to a mammalian patient, the
stem cell conditioned medium comprising a cell culture supernatant
containing at least one factor capable of exerting effective
neuroprotection to treat a mammalian neural injury or insult.
Inventors: |
March; Keith Leonard;
(Carmel, IN) ; Johnstone; Brian H.; (Indianapolis,
IN) ; Du; Yansheng; (Westfield, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
March; Keith Leonard
Johnstone; Brian H.
Du; Yansheng |
Carmel
Indianapolis
Westfield |
IN
IN
IN |
US
US
US |
|
|
Family ID: |
39107733 |
Appl. No.: |
14/018849 |
Filed: |
September 5, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13236566 |
Sep 19, 2011 |
|
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14018849 |
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Current U.S.
Class: |
424/574 |
Current CPC
Class: |
A61K 35/28 20130101;
A61P 25/16 20180101; C12N 2502/1305 20130101; C12N 5/0667 20130101;
C12N 2502/00 20130101; C12N 2502/1382 20130101; C12N 5/0668
20130101; C12N 2502/1352 20130101; A61P 25/28 20180101; C12N 5/0663
20130101; C12N 5/0619 20130101; A61P 25/00 20180101; A61P 25/08
20180101; C12N 2502/1358 20130101; C12N 5/06 20130101; A61K 35/35
20130101; A61P 29/00 20180101 |
Class at
Publication: |
424/574 |
International
Class: |
A61K 35/12 20060101
A61K035/12 |
Claims
1. A method of treating a mammalian patient having a neuronal
injury or insult, the method comprising the step of: administering
a therapeutically-effective dose of an adipose tissue-derived stem
cell conditioned medium to a mammalian patient, the stem cell
conditioned medium comprising a cell culture supernatant containing
at least one factor obtained from at least one adipose
tissue-derived stem cell, the at least one factor capable of
exerting effective neuroprotection to treat a mammalian neural
injury or insult.
2. The method of claim 1, wherein the neural injury or insult is an
acute injury selected from the group consisting of an acute
cerebral hypoxia, an acute spinal cord injury, an acute brain
injury, and acute inflammation.
3. The method of claim 1, wherein the neural injury or insult is a
central nervous system injury selected from the group consisting of
a brain injury and a spinal cord injury.
4. The method of claim 1, wherein the neural injury or insult
comprises a neurodegenerative disorder.
5. The method of claim 1, wherein the neural injury or insult
comprises a chronic injury.
6. The method of claim 5, wherein the chronic injury is selected
from the group consisting of Parkinson's disease, Alzheimer's
disease, amyotrophic lateral sclerosis, multiple sclerosis,
cerebral palsy, peripheral neuropathy, Huntington's disease,
epilepsy, encephalomyelitis, encephalitis, a chronic spinal cord
injury, a chronic brain injury, and chronic cerebral hypoxia.
7. The method of claim 1, wherein the neural injury or insult
comprises a hypoxia-ischemia of the brain selected from the group
consisting of neonatal cerebral hypoxia and adult cerebral
hypoxia.
8. The method of claim 1, wherein the step of administering is
performed by administering a therapeutically effective dose of a
stem cell conditioned medium to a mammalian patient selected from
the group consisting of a neonatal patient, a child, an adolescent,
and an adult patient.
9. The method of claim 1, wherein the step of administering is
performed by systemically administering a therapeutically effective
dose of a stem cell conditioned medium to a mammalian patient.
10. The method of claim 9, wherein the systemic administration is
selected from the group consisting of injection administration and
intravenous administration.
11. The method of claim 1, wherein the step of administering is
performed by locally administering a therapeutically effective dose
of a stem cell conditioned medium to a mammalian patient at or near
a site of neural injury or insult.
12. The method of claim 11, wherein the local administration is
selected from the group consisting of interarterial administration,
intravenous intraparenchymal administration, intrathecal
administration, and interperitoneal administration.
13. The method of claim 1, wherein the step of administering is
performed to treat a mammalian neural injury or insult by treating
neural cells in vivo that are at risk for hypoxia induced neuronal
death.
14. The method of claim 1, wherein the step of administering is
performed to treat a mammalian neural injury or insult by treating
cerebral tissues damaged by hypoxia.
15. The method of claim 1, wherein the step of administering is
performed to treat a mammalian neural injury or insult by
stimulating neural cell regeneration.
16. The method of claim 1, wherein the cell culture supernatant is
produced by culturing at least one mammalian adipose stem cell to
produce the at least one factor.
17. A method of treating a mammalian patient having a neuronal
injury or insult, the method comprising the step of: administering
a therapeutically-effective dose of an adipose tissue-derived stem
cell conditioned medium to a mammalian patient, the stem cell
conditioned medium comprising a cell culture supernatant containing
at least one factor obtained from at least one adipose
tissue-derived stem cell, the at least one factor capable of
exerting effective neuroprotection to treat a chronic mammalian
neural injury or insult.
18. A method of treating a mammalian patient having a neuronal
injury or insult, the method comprising the step of administering a
therapeutically-effective dose of an adipose tissue-derived medium
containing at least one factor from a mammalian stem cell to a
mammalian patient, the at least one factor from the mammalian stem
cell capable to treat a mammalian neural injury or insult.
19. The method of claim 18, wherein the mammalian stem cell is from
a human patient, and wherein the mammalian patient comprises the
human patient, and wherein the mammalian stem cell was obtained
from the human patient.
20. The method of claim 18, wherein the mammalian stem cell is from
an animal patient, and wherein the mammalian patient comprises the
animal patient, and wherein the mammalian stem cell was obtained
from the animal patient.
Description
PRIORITY
[0001] The present application is related to, claims the priority
benefit of, and is a U.S. continuation application of, U.S.
Nonprovisional patent application Ser. No. 13/236,566, filed Sep.
19, 2011, which is related to, claims the priority benefit of, and
is a U.S. continuation application of, U.S. Nonprovisional patent
application Ser. No. 11/844,941, filed Aug. 24, 2007 and issued as
U.S. Pat. No. 8,021,882 on Sep. 20, 2011, which is related to, and
claims the priority benefit of, U.S. Provisional Patent Application
Ser. No. 60/823,460, filed Aug. 24, 2006. The contents of each of
these applications are hereby incorporated by reference in their
entirety into this disclosure.
BACKGROUND
[0002] Pluripotent cells, sometimes referred to as stem cells, are
characterized by an ability to differentiate into a variety of
different cells. Some pluripotent cells types, such as human
embryonic stem cells, display an ability to differentiate into the
broadest spectrum of cells; in fact, embryonic stem cells display
an ability to differentiate into practically any type of cell that
exists within the human tissues. However, as embryonic stem cells
develop and differentiate into lines of partially and/or fully
differentiated cells, those further differentiated cells lose some
or all of their pluripotent ability because embryonic stem cells
have the ability to "morph" into practically any cell type, the
scientific community has explored the possibility of using these
embryonic stem cells to replace those injured or dying cells in
individuals suffering neurodegenerative disease such as Parkinson's
disease.
[0003] However, embryonic stem cells have limitations in their
ability to be used clinically, as they must be derived from another
individual--an embryo. This not only raises a potential that the
patient will reject the cells, but it also severely limits the
ability for such cells to be used in the first place. Therefore,
much effort has been made in finding pluripotent cells that are
obtainable in large quantities, that can differentiate into a
target cell, and that will not be rejected by the individual being
treated thereby. One such pluripotent cell that has been used for
autologous cell therapy to regenerate neural tissue is the
pluripotent cells found in the "stromal" or "non-adipocyte"
fraction of the adipose tissue. These pluripotent cells were
previously considered to be pre-adipocytes, i.e. adipocyte
progenitor cells (hereinafter "adipose stem cells" or "ASC"). Zuk,
2001. Data suggests that these adipose stem cells have a wide
differentiation potential, as research by Zuk using subcutaneous
human ASCs in vitro were able to be differentiated into adipocytes,
chondrocytes and myocytes. Id. Further studies by Erickson et al.,
showed that human ASCs could differentiate in vivo into
chondrocytes following transplantation into immune-deficient mice,
and studies by Stafford showed that human ASCs were able to
differentiate into neuronal cells. (Erickson, 2002); (Stafford,
2002). More recently, it was demonstrated that human ASCs were able
to differentiate into neuronal cells, osteoblasts (Dragoo, 2003),
cardiomyocyte (Rangappa, 2003; Planat-Benard, 2004), and
endothelial cells (Planat-Benard, 2004). As such studies suggest
that the delivery of certain pluripotent cells to neural tissue
damaged by stroke or cardiovascular disease may cause regeneration
of the damaged tissue through differentiation of the delivered
pluripotent cells.
[0004] Therefore, treatments using autologous pluripotent cells,
and ASCs in particular, have necessarily centered upon the harvest
and concentration of the pluripotent cells from a remote area of
the patient to be treated, followed by application of those
concentrated pluripotent cells to an injured or targeted site so
that the pluripotent cells can differentiate and take the place of
the damaged cells at the target. See, e.g., U.S. Pat. No. 7,078,230
to Wilkison et al.; U.S. Patent App. Pub. No. U.S. 2005/0260174 to
Fraser et al.
BRIEF SUMMARY
[0005] At least embodiment of the present disclosure provides
harvesting adipose stem cells (ASC) recovered from adipose tissue
to provide an autologous source of cells. These pluripotent cells,
which reside in the "stromal" or "non-adipocyte" fraction of the
adipose tissue, have the capacity to differentiate in culture into
adipocytes, chondrocytes, osteoblasts, neuronal cells, and
myotubes. ASCs can be obtained in large quantities, in the range of
10.sup.8 to 10.sup.9 cells, following routine liposuction of
subcutaneous adipose tissue. The ready accessibility of these cells
provides for a particularly feasible and attractive form of cells
for harvest and presents the opportunity to retrieve a given
patient's own cells as a source of pluripotent cells for
harvesting.
[0006] Another embodiment of the present disclosure relates to the
use of harvested ASCs or other stem cells to secrete bioactive
levels of therapeutic proteins that can promote repair of injured
and diseased neural tissues or prevent neural tissue death under
circumstances that would ordinarily result in apoptosis. According
to one aspect, still another embodiment relates to using media
exposed to ASCs maintained and/or growing in cell culture to
produce a composition that promote the growth, health, protection,
and/or development of various types of human and animal cells,
especially neural cells.
[0007] Yet another embodiment of the present disclosure relates to
the use of at least one factor produced by ASCs to effect changes
in other cells exposed to the factors. In one embodiment, at least
one factor produced by an ASC is used to prevent the death of
neuronal cells either in culture or in the central or peripheral
nervous system of an adult or developing animal, including Homo
Sapiens.
[0008] Still another embodiment of the present disclosure related
to a method of treating diseases, disorders or injuries in neural
tissue by exposing neuronal tissues and various cells therein to
products produced by ASCs.
[0009] Still another embodiment of the present disclosure relates
to using media exposed to ASCs to modulate physiological processes
such as formation of new vessels or expansion of existing vessels
within the central nervous system, peripheral nervous system, or
spinal cord. At least one embodiment relates to a method of
treating at least a portion of the central nervous system
comprising the steps of administering at least a fractionated
portion of the media exposed to ASC to at least one region of the
Central Nervous System in either a human or animal patient.
[0010] At least one embodiment of the present disclosure relates to
a method of using substances secreted by ASCs into the cell culture
medium to modulate at least one of several in vitro neuronal injury
pathways.
[0011] Still another embodiment of the present disclosure relates
to recovery of at least one compound produced by ASCs in cell
culture to a cell culture media in contact with ASCs to modulate
the activity of neuronal cells.
[0012] Yet another embodiment of the present disclosure relates to
using at least substance derived from ASC media cultured in vitro
to treat cells in vivo at risk for ischemia-hypoxia-induced
neuronal death.
[0013] Another embodiment of the present disclosure relates to the
in vitro use at least one of substances derived from ASC cells
cultured in vitro to affect the activity, viability and/or
differentiation of neuronal cells either in vivo or in vitro.
[0014] Still another embodiment of the present disclosure is a
method of treating hippocampal tissue that has been damaged by
neonatal hypoxia-ischemia comprising the steps of providing at
least substance or compound derived from media recovered from
cultures of ASCs and administering at least one dosage of the at
least one factor to hippocampal neuron tissue and/or cells.
[0015] In at least one embodiment of a method of treating a
mammalian (human or animal) patient having a neuronal injury or
insult of the present disclosure, the method comprises the step of
administering a therapeutically-effective dose of a stem cell
conditioned medium to a mammalian patient, the stem cell
conditioned medium comprising a cell culture supernatant containing
at least one factor capable of exerting effective neuroprotection
to treat a mammalian neural injury or insult. In another
embodiment, the neural injury or insult is selected from the group
consisting of a chronic injury, an acute injury, a central nervous
system injury, and a peripheral nervous system injury. In yet
another embodiment, the acute injury is selected from the group
consisting of a cerebral hypoxia, a spinal cord injury, a brain
injury, and inflammation. In an additional embodiment, the central
nervous system injury is selected from the group consisting of a
brain injury and a spinal cord injury.
[0016] In at least one embodiment of a method of treating a
mammalian patient having a neuronal injury or insult of the present
disclosure, the neural injury or insult comprises a
neurodegenerative disorder. In an additional embodiment, the
neurodegenerative disorder is selected from the group consisting of
Parkinson's disease, Alzheimer's disease, amyotrophic lateral
sclerosis, multiple sclerosis, cerebral palsy, peripheral
neuropathy, Huntington's disease, epilepsy, encephalomyelitis,
encephalitis, a spinal cord injury, a brain injury, and cerebral
hypoxia. In yet an additional embodiment, the neural injury or
insult comprises a hypoxia-ischemia of the brain selected from the
group consisting of neonatal cerebral hypoxia and adult cerebral
hypoxia. In another embodiment, the step of administering is
performed by administering a therapeutically effective dose of a
stem cell conditioned medium to a mammalian patient selected from
the group consisting of a neonatal patient, a child, an adolescent,
and an adult patient.
[0017] In at least one embodiment of a method of treating a
mammalian patient having a neuronal injury or insult of the present
disclosure, the step of administering is performed by systemically
administering a therapeutically effective dose of a stem cell
conditioned medium to a mammalian patient. In another embodiment,
the systemic administration is selected from the group consisting
of injection administration and intravenous administration. In yet
another embodiment, the step of administering is performed by
locally administering a therapeutically effective dose of a stem
cell conditioned medium to a mammalian patient at or near a site of
neural injury or insult. In an additional embodiment, the local
administration is selected from the group consisting of
interarterial administration, intravenous intraparenchymal
administration, intrathecal administration, and interperitoneal
administration.
[0018] In at least one embodiment of a method of treating a
mammalian patient having a neuronal injury or insult of the present
disclosure, the step of administering is performed to treat a
mammalian neural injury or insult by treating neural cells in vivo
that are at risk for hypoxia induced neuronal death. In an
additional embodiment, the step of administering is performed to
treat a mammalian neural injury or insult by treating cerebral
tissues damaged by hypoxia. In yet an additional embodiment, the
step of administering is performed to treat a mammalian neural
injury or insult by stimulating neural cell regeneration. In
another embodiment, the cell culture supernatant is produced by
culturing at least one mammalian adipose stem cell to produce the
at least one factor.
[0019] In at least one embodiment of a method of treating a
mammalian patient having a neuronal injury or insult of the present
disclosure, the method comprises the step of administering a
therapeutically-effective dose of a stem cell conditioned medium to
a mammalian patient, the stem cell conditioned medium comprising a
cell culture supernatant containing at least one factor capable of
exerting effective neuroprotection to treat a mammalian neural
injury or insult selected from the group consisting of a chronic
injury, an acute injury, a central nervous system injury, a
peripheral nervous system injury, and a neurodegenerative
disorder.
[0020] In at least one embodiment of a method of treating a
mammalian patient having a neuronal injury or insult of the present
disclosure, the method comprises the step of administering a
therapeutically-effective dose of a medium containing at least one
factor derived from a mammalian stem cell to a mammalian patient to
treat a mammalian neural injury or insult. In another embodiment,
the mammalian stem cell comprises a human mammalian stem cell, and
wherein the mammalian patient comprises a human patient, and
wherein the human mammalian stem cell was obtained from the human
patient. In yet another embodiment, the mammalian stem cell
comprises an animal mammalian stem cell, and wherein the mammalian
patient comprises an animal patient, and wherein the animal
mammalian stem cell was obtained from the animal patient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1: Phase contrast micrographs of (A) human, (B) mouse,
and (C) rat grown in EGM2MV.
[0022] FIGS. 2A and 2B: In vitro expansion of (FIG. 2A) human and
(FIG. 2B) mouse ASCs.
[0023] FIG. 3: Profile of cytokines expressed by human ASCs
cultured under normoxia (A) or hypoxia (B). Conditioned media was
applied to a RayBio antibody array VII and developed for
visualization. Numbers refer to antibodies (in duplicate) for
specific proteins. A "+" or "-" sign signifies positive or negative
controls, respectively. The proteins (with detection limits in
pg/ml) are: 1, angiogenin (10); 2, MCP-1 (3); 3, IL-1ralpha (10);
4, IL-5 (1); 5, IL-6 (1); 6, MIP-3a (100); 7, SCF (10); and 8,
TNF-beta (1000).
[0024] FIG. 4: Influence of ASC-Conditioned Medium on endothelial
cell ec Survival and prevention of apoptosis.
[0025] FIGS. 5A, 5B, and 5C: Conditioned media from ASCs (stromal
cell-conditioned medium: ASC Conditioned Media) protects CGN
neurons against potassium (5 mM)-induced apoptosis. The rat ASCs
were cultured in EGM2MV media to confluence, and then switched into
basal media Eagle (BME with 5 mM K.sup.+, Invitrogen) for 24 hours.
The conditioned media (ASC Conditioned Media) were collected from
the ASCs culture and subsequently added to the regular BME (5 mM
K.sup.+) at volumes equivalent to 30% of the total media volume.
The BME with ASC Conditioned Media was then added to rat CGN
cultures. Viable neurons was quantified by counting fluorescein
(green) positive cells which result from the de-esterification of
fluorescein diacetate (FDA, Sigma, 10 .mu.g/ml, 5 min) by living
cells. Propidium iodide (PI, Sigma, 5 .mu.g/m, 5 min), which
interacts with nuclear DNA of dead cells, producing a red
fluorescence, was used to identify dead neurons (Du, 1997a). FIG.
5A) untreated CGN (control). FIG. 5B) cultures exposed to LK (5 mM)
BME for 24 h. FIG. 5C) cultures exposed to LK (5 mM) media with 30%
ASC Conditioned Media for 24 h. Data are from a representative
experiment repeated twice with similar results.
[0026] FIG. 6: ASC Conditioned Media protects CGN neurons against
LK-induced apoptosis in a dose-dependent fashion. The rat ASC
Conditioned Media (2--30% of final vol) was added to the rat CGN
cultures following LK treatment. The cultures were then double
stained with FDA and PI (as demonstrated in FIG. 5). Data are from
a representative experiment repeated twice with similar
results.
[0027] FIG. 7: ASC Conditioned Media protects CGN neurons against
glutamate (50 .mu.M)-induced neuronal death. The ASC Conditioned
Media was collected and subsequently added into the CGN cultures
(30% or 50% replacements) that were then challenged by 50 .mu.M of
glutamate. Neuronal viability was quantified by staining neurons
with FDA (Du, 1997). G indicates that the CGN were exposed to 50
.mu.M glutamate; SCM+G, CGN exposed to 50 .mu.M glutamate and the
indicated percentages of ASC Conditioned Media.
[0028] FIG. 8: ASC Conditioned Media collected from fresh (P0) or
passage 3 (P3) ASCs protects CGN neurons against glutamate (50
.mu.M)-induced neuronal death differently. The 50% replacement of
P0 ASC Conditioned Media exerts a stronger neuroprotective effect
than P3 ASC Conditioned Media. Neuronal viability was quantified by
staining neurons with FDA (Du, 1997). Control is CGN without 50
.mu.M glutamate exposure; G indicates that the CGN were exposed to
50 .mu.M glutamate; SCMP0+G and SCMP3+G, CGN exposed to 50 .mu.M
glutamate and 50% ASC Conditioned Media from cells at P0 or P3,
respectively.
[0029] FIG. 9: ASC Conditioned Media collected from fresh ASCs has
been enriched 50.times. using 10K CentriPlus protects CGN neurons
against glutamate (50 .mu.M)-induced neuronal death. Addition of
250.times. enriched ASC Conditioned Media almost completely
protects neurons against glutamate toxicity in CGN. Neuronal
viability was quantified by staining neurons with FDA (Du, 1997). G
indicates that the CGN were exposed to 50 .mu.M glutamate; G+CSCM
CGN exposed to 50 .mu.M glutamate and the indicated percentages of
250.times. Concentrated ASC Conditioned Media.
[0030] FIG. 10: Glutamate treatments induced JNK and p38
phosphorylation in CGN. Immunoblot analyses were performed with
antibodies against phosphorylated JNK and p38 (p-JNK and pp38), and
p38 (Santa Cruz). Glutamate treatments increase p-JNK and pp38 by 3
h post-treatment. Note that glutamate treatment fails to alter
total p38 expression in the same samples. C=control (no glutamate
treatment). Glu=glutamate treatment. Each condition represents 3
samples.
[0031] FIG. 11: ASC Conditioned Media collected from P3 human ASCs
(HASC Conditioned MediaP3) protects CGN neurons against glutamate
(50 .mu.M)-induced neuronal death. The 50% replacement of P3 HSMC
exerts a strong neuroprotective effect on rat CGN followed by
glutamate treatments. Neuronal viability was quantified by staining
neurons with FDA (Du, 1997). Control, CGN without 50 .mu.M
treatment; G, CGN exposed to 50 .mu.M glutamate; 50% HSCMP3+G, CGN
exposed 50 .mu.M glutamate and 50% ASC Conditioned Media.
[0032] FIG. 12: ASC Conditioned Media protects CGN neurons against
H.sub.2O.sub.2 (50 .mu.M)-induced neuronal death. The ASC
Conditioned Media was collected and subsequently added into the CGN
cultures (at 1/3 of the final volume) that were then challenged by
adding 50 .mu.M of H.sub.2O.sub.2. Neuronal viability was
quantified by staining neurons with MTT (Du, 2003). C, untreated
CGN; H202 50 .mu.M, CGN exposed to 50 .mu.M H.sub.2O.sub.2 H202+SCM
30%, CGN exposed to 50 .mu.M H.sub.2O.sub.2 and 30% ASC Conditioned
Media; STM 30%, CGN exposed to 30% ASC Conditioned Media only.
[0033] FIG. 13: ASC Conditioned Media treatment improved cortical
neuron survival following oxygen and glucose deprivation (OGD)
injury. ASC Conditioned Media (added to a final concentration of
5%-100% by replacing the medium) protects cortical neurons in a
dose-dependent fashion. SCM, ASC Conditioned Media added at the
indicated percentages.
[0034] FIGS. 14a, 14b, and 14c: ASC Conditioned Media prevents
neuronal loss when administered to neonatal rats at 24 hours
following hypoxic-ischemic injury. Representative coronal sections
of postnatal day 14 (P14) rat brains demonstrate that 7 days
following unilateral (left) carotid ligation and exposure to
hypoxia (7%) for 2 hours at P7 (Wei, 2004) with or without
treatment with ASC Conditioned Media (10 .mu.l/pup, 24 h following
H-I treatments). FIG. 14a. normal, FIG. 14b. ischemia-treated with
BME, FIG. 14c. ischemia treated following 24 h post treatment with
ASC Conditioned Media. Note the moderate damage in the hippocampus
ipsilateral to carotid ligation in animals (compare FIG. 14a vs.
FIG. 14b) and the significant protection by ASC Conditioned Media
(FIG. 14b vs. FIG. 14c). Rectangle indicates the lesion site in the
left hippocampus. The area of tissue in the hippocampus ipsilateral
to the lesioned hemisphere was compared in the same animal with the
area of tissue remaining in the matching brain region contralateral
to unlesioned hemisphere. The percentage area loss was then
determined in each animal, and data are presented as the mean plus
or minus SEM for each group. ASC Conditioned Media 24 hours after
hypoxia "significantly" protects against hippocampal volume loss
induced by ischemic injury (73.+-.3 vs. 90.+-.1, n=2/group, one-way
ANOVA, * p<0.05).
[0035] FIG. 15: Results of proteomic analysis of ASC Conditioned
Media recovered from media contacted with ASCs in vitro. Specific
proteins identified in this experiment include, but are not limited
to, proteins related to neuroprotection: NGF (nerve growth factor),
GDNF (glia-derived neurotrophic factor, IGF-1 (Insulin-like growth
factor, and VEGF (vascular endothelia growth factor).
[0036] FIG. 16: As expected, in inactivation of a single factor
present in ASC Conditioned Media, which is known to limit damage
due to a specific insult, significantly reduces the ability of ASC
Conditioned Media to protect against the specific damaging agent.
Injury was induced in CGN by exposure to 40 .mu.M glutamate as
described above. ASC Conditioned Media, protected the CGN from
damage (G+ASC). Pretreatment of ASC Conditioned Media with an
anti-BDNF antibody that neutralizes BDNF activity significantly,
but not totally, reduced the ability of ASC Conditioned Media to
protect CGN (G+antiBDNF+ASC). Conversely, it would not be expected
that BDNF activity would protect neutrons from injury due to other
damaging agents or conditions, such as OGD, oxidation (e.g.,
H.sub.2O.sub.2), or toxins (E.g., MPP).
[0037] FIG. 17 FIGS. 17a and 17b: Results of tests showing that
ASC-CM preserves the cognitive function of rats following
hypoxia-ischemia injury, and utilizing the Morris Water Maze
test.
[0038] FIG. 18 displays the results of an experiment indicating
that ASC-CM protects neurons against 6-hydroxydopamine
(6-OHDA)-mediated death.
[0039] FIG. 19 displays the results of PC12 cells were cultured in
DMEM containing 10% FBS for 3 days, then starved in BME medium
without FBS for 24 hours. Various percentages of the medium was
exchanged for an equivalent volume of ASC-CM, as indicated in FIG.
19.
DETAILED DESCRIPTION
[0040] For the purposes of promoting an understanding of the
principles of the disclosure, reference will now be made to the
embodiments illustrated herein and specific language will be used
to describe the same. It will nevertheless be understood that no
limitation of the scope of the disclosure is thereby intended.
[0041] As used herein, a therapeutically effective dosage or amount
of a compound is an amount sufficient to affect a positive effect
on a given medical condition. The affect, if not immediately, may,
over period of time, provide a noticeable or measurable effect on a
patient's health and well being.
[0042] According to one aspect of the present disclosure, it has
been found that when ASCs are cultured in vitro, the ASCs secrete a
combination of angiogenic and antiapoptotic factors and/or
additional compounds (either as single factors, or in combination
with one another) in relative concentrations and combinations that
have been shown to exert effective neuroprotection in a variety of
mechanistically distinct neuronal death pathways. As a result,
according to one embodiment, a therapeutically effective dose of
ASC secretions from in vitro culture is administered to prevent or
counteract a chronic or acute neural injury or insult.
[0043] In particular, according to at least one embodiment, it was
found that media used to culture and/or maintain ASCs in vitro has
the unexpected characteristic of protecting neural tissue and/or
encouraging regeneration from stimuli-induced damage when
administered to a patient in a therapeutically effective dosage.
This media, or extracts thereof, appears to inhibit critical
neuronal death pathways due to the presence of several
complementary neuroprotective factors which combine to limit
neuronal death and/or stimulate regeneration of neural cells in
vitro as well as in vivo in the context of neural insult, although
the mechanism is not entirely understood. Further, fractions of
media conditioned by ASCs are a source of various factors that
either alone or in combination with one another have shown an
ability to affect neuronal cells that are subject to acute or
chronic injury. Accordingly, the ASC conditioned media has been
shown to be an excellent source of material useful for producing,
concentrating, and isolating a broad spectrum combination of
compounds in relative percentages and forms that effect a
therapeutic effect when administered to an individual suffering
from a neural insult, including central nervous system or
peripheral nervous system injuries that may be chronic or acute in
nature. Further, ASC conditioned media has been shown to be an
excellent source of material useful for producing, concentrating,
and isolating individual compounds, or groups of compounds, that
have been shown to protect neuronal cells from death, damage and
insult and/or to cause regeneration thereof.
[0044] For example, the ASC conditioned media and/or fractions and
concentrations thereof have proven effective to treat or prevent
various disorders that involve hypoxia-ischemia (H-I) of the brain
including neonatal or adult H-I-induced encephalopathy, stroke and
neurodegenerative disorders, and such treatments do not carry the
same risk of rejection as that shown by injecting foreign stem
cells into a patient, since at least one embodiment does not inject
any cells whatsoever into a patient. Further, application of ASC
conditioned media and/or fractions and concentrations thereof can
be used to treat chronic or acute injuries in the peripheral
nervous system, central nervous system, and/or spinal cord in
either neonatal individuals, children, or adults.
[0045] It will be appreciated that other stem cells or pluripotent
cells may be utilized, such as other mesenchymal stem cell (MSC),
which are found in the stroma of different tissues throughout the
body. Human MSCs (including ASCs) are characterized by the surface
marker profile of lin-/CD45-/c-kit-/CD90+. Further, in one
embodiment, appropriate stem cells display the CD34+ positive at
the time of isolation, but lose this marker during culturing.
Therefore the full marker profile for one stem cell type that may
be used according to the present application is
lin-/CD45-/c-kit-/CD90+/CD34. In another embodiment utilizing mouse
stem cells, the stem cells are characterized by the Sca-1 marker,
instead of CD34, to define what appears to be a homologue to the
human cells described above, with the remaining markers remaining
the same. It will be appreciated that other stem cells with similar
marker profiles could be used, such as the pluripotent stem cell
from skeletal muscle that was identified by Case et al (Annals of
NY Acad. Sci. vol. 1044:183-200). Case indicates that these cells
appear to exist in the adipose and brain theorize that these are
the same stem cell that resides in many different organs. They have
termed these cells common pluripotent stem cells (CoPSC), but other
stem cells could be used, with the exception of hematopoietic stem
cells. This would also include progenitor cells that derive from
MSCs.
[0046] The process for producing the ASC conditioned media is
further outlined below, as is the method for using the media or its
fractions or distillates to treat nervous system insults or
neurodegenerative conditions. While the term "ASC conditioned
media" is used throughout, it is understood that the same processes
will apply to production of similar media through other stem cells,
but that adipose stem cells are used for exemplary purposes.
I. Process for Producing ASC-Conditioned Media
[0047] A. Isolation and Expansion of Adipose Stem Cells
[0048] To isolate human ASCs, lipoaspirates (250-500 ml) were
obtained from patients undergoing elective liposuction procedures
and processed essentially according to Zuk et al. ASCs were plated
at 1,000-10,000 cells/cm.sup.2. Most ASCs attach to the flask and,
when cultured in EGM2MV media for example, can be multiplied
50-fold in 8 days (FIGS. 1 and 2) with their growth rate decreasing
when they reach confluency. FIG. 2B demonstrates the expansion of
murine ASCs following their isolation using similar protocols.
Similar growth data has been obtained with rat ASCs as shown in
FIG. 1C).
[0049] These experiments demonstrate that ASCs are readily isolated
and rapidly expanded ex vivo from relatively small amounts of
adipose tissue, thus laying the groundwork for using autologous
ASCs in research and clinical settings. As discussed further below,
neuroprotective studies suggest that 30% of culture media replaced
by the culture media from 10.sup.8 to 10.sup.9 autologous cells
exerts significant neuroprotective effects on different
neurodegenerative models.
[0050] B. Preparation of ASC-Conditioned Media
[0051] The ASCs were cultured in EGM2MV medium to confluence, and
then switched into growth-factor free basal media (EBM-2,
Clonetics) for conditioning in either normoxic or hypoxic
conditions for 72 hours. Cell supernatants were collected and
subsequently assayed for cytokines using a RayBio array VI and VII.
FIG. 3 displays the findings of array VII, and indicate that in
addition to the pluripotency of ASCs, the endocrine or paracrine
potential of ASCs may have significant therapeutic relevance.
Testing as shown that ASCs delivered to the CNS in the setting of
degeneration as a result of ischemia due to stroke in this case may
be able to protect neurons from death processes as well as enhance
angiogenesis by both differentiating into vascular phenotypes, and
by recruiting resident mature vascular endothelial cells to
integrate into the nascent vascular network. More importantly, in
experiments directed to determining the overall biological effect
of the ASC conditioned media, human microvascular endothelial cells
were exposed to the media conditioned by ASCs incubated in (1)
normoxic conditions, (2) hypoxic conditions, and (3) unconditioned
media as a control. As shown in FIG. 4, after 4 days, exposure to
ASC-normoxic media resulted in an 80% increase in HMVEC number,
while the ASC-hypoxic media resulted in a 160% increase in HMVEC
number as compared to the control. These significant increases
confirm the potential of ASC conditioned media to promote growth or
survival of other cells in their vicinity.
[0052] C. Isolation of Components in ASC Conditioned Media by
Fractionation
[0053] Our studies illustrate that ASCs Conditioned Media during
culture plays a critical role in protecting neurons from injurious
stimuli. Defined protein fractions (<10 k, 10-30 k, 30-50 k, and
>50 k) from the conditioned media have been evaluated with
respect to their ability to block neuronal death in specific,
well-defined in vitro models. Data shows that the passage number of
the ASCs as well as various environmental stimuli influence the
level and composition of factor secreted into the media and the
resulting neuroprotective efficacy of conditioned media.
[0054] Neuronal death is mediated by complementary neuronal death
pathways in distinct neurodegenerative models, and may be limited
in each model by distinct trophic factor(s) by ASCs within the
conditioned media. Neuroprotective factors the conditioned medium,
such as VEGF and HGF, can be selected for by selectively altering
the activity of present factors through the addition of
inactivating antibodies, or conversely, by adding purified
recombinant proteins to fractionated supernatants.
[0055] Evaluation of the Neuroprotective Capacity of Different
Fractions of Media from ASCs in Different In Vitro Neuronal Death
Models.
[0056] Factors secreted into the media during culture of ASCs
potently protect neurons from injury stimuli associated with
specific neuronal death pathways. Defined fractions of conditioned
media from human, murine and rat ASCs, at different passages and
subjected to hypoxia or normoxia, have been enriched by >50-fold
in order to evaluate increase the potency of neuroprotection and to
enable detection of low abundance proteins by proteomic assays.
Once the conditioned medium is enriched, it is optionally
fractionated by, for example, size exclusion and then can be
concentrated. Optionally, ASC Conditioned Media is simply
concentrated and/or fractionated. In this manner, concentrations of
the ASC Conditioned Media can be manipulated.
[0057] According to another embodiment, ASCs derived from human,
mouse or rat adipose tissue, as described above are evaluated by
flow cytometry to confirm that they are CD34+, CD90, c-kit-, CD31-,
CD45- and, in the case of murine cells, Sca1+. The cells are then
plated in DMEM/F12 with 10% FBS with no additional growth factors
added; and EGM/2MV (Cambrex) is used. The resulting ASC Conditioned
Media is thereafter applied for treatment or prevention of neural
injury.
[0058] ASC Conditioned Media is enriched and size fractionated
supernatants can be further fractionated using a centrifugal filter
device (10K, 30K, and 50K Centriplus, Amicon, Mass.). Centriplus
molecular weight filters can provide an 100-fold sample enrichment
and can easily be used to enrich samples by 50 to 250-fold using a
10 kDa. Neuroprotective factors in different fractions of the
supernatant are likely to be predominantly within the ranges of
<10 kDa, 10-30 kDa, 30-50 kDa, and >50 kDa. It is known that
growth factors, for example, have a large size difference (such as
GCSF 20 kDa BDNF 27 kDa EPO 34 kDa VEGF 45 kDa, and NGF 116 kDa)
based on size separation and will segregate in different molecular
weight fractions. Optional steps include collecting and
fractionating supernatants from fresh or passaged ASCs grown under
normoxic or hypoxic conditions, and using these fractions for
neuroprotection in LK/HK, glutamate, H2O2, ODG, 60HDA, and MPP+CGN
models.
[0059] D. Delivery, Timing, and Dosing of ASC Conditioned Media
[0060] 1. Delivery
[0061] Unlike cell therapies that inject stem cells at the point of
injury, the process for treatment of injured nervous system cells,
or cells prone to injury or neurodegenerative diseases does not
require localized injection. Rather, it will be appreciated that
since no living cells, which may die if used systemically, are
being utilized, that a wide array of delivery systems may be used
to ensure that the ASC conditioned media, its fractions,
concentrations, or distillations may be delivered systemically, via
injection, intravenously, or otherwise. Optionally, the ASC
conditioned media may be delivered locally at the site of injury.
For example, the ASC Conditioned Media may be delivered
interarterially, intravenously intraparenchemally, intrathecally,
or interperitonally.
[0062] 2. Timing
[0063] In certain models, such as in the H-I model, that at 24 h
following induction of hypoxia, the Blood Brain Barrier ("BBB") is
disrupted, allowing peptide penetration. Additionally, some growth
factors, such as GCSF and IGF-1, can penetrate into the brain
immediately after H-I treatment. Identifying key factors for
neuroprotection, initially concentrated conditioned ASC Conditioned
Media were used. 7 day old pups underwent will undergo H-I,
followed by iv injections of 1-10 .mu.l of 250-fold concentrated
rat ASC conditioned supernatant fractions or a cocktail with
defined growth factors at 2, 8 and 24 hours post surgery. As a
control, animals were injected with the same amount of BME
media.
[0064] The first few hours following H-I are believed to be the
most critical for neuronal death resulting from direct effects of
the insult. Secondary damage, triggered by inflammation, occurs
after 48 hours. Given the prolonged period in which damage occurs,
it may be beneficial to repeat dosing in order to effectively block
neuronal death. Additionally, the ASC conditioned media or fraction
thereof may have function to regenerate neurons derived from stem
cells. Accordingly, the ASC Conditioned Media or fraction thereof
is optionally administered at an optimized concentrate at least
once daily for at least one day, at least 2 days, or at least 3
days following insult (such as H-I insult, onset of
neurodegeneration, or surgery). Further optionally, due to the
neural protection shown when ASC conditioned media or a fraction
thereof is administered prior to insult, the ASC conditioned media
may be optionally administered at least one day, at least 2 days,
or at least 3 days prior to the insult or surgery.
[0065] 3. Dosage
[0066] According to one aspect of the present application, a
therapeutic dose of the ASC conditioned is delivered to an
individual. In one embodiment, a defining characteristics of the
ASC conditioned media are naturally-derived factors secreted into
the medium during fermentation of ASCs. This conditioned medium
(CM) possesses the qualities of being able to prevent damage to
neurons due (a) ischemic events, (b) induction of cell-death
processes (apoptosis), (c) exocitotoxity, (d) oxidation, or (e)
neuron-specific damaging agents. In vitro assays for each would be
(a) oxygen-glucose deprivation (OGD), (b) Low K model, (c)
glutamate exocitoxicity, (d) hydrogen peroxide, and (e)
6-hydroxy-dopamine toxicity of dopaminergic neurons at a
therapeutic dose.
[0067] According to one embodiment, the ASC-CM is concentrated at
least 50 fold, at least 100-fold, at least 200 fold, or at least
1000-fold. Optionally, the concentrated ASC-CM is fractionated
through a size exclusion resin or membrane to remove substances
less than 5 kDa, less than 10 kDa, less than 20 kDa, less than 30
kDa, less than 40, kDa, or less than 50 kDa. The concentrated
ASC-CM is then optionally stabilized to protect degradation or loss
of components. According to one exemplary embodiment of dosing, 800
MICROLITERS/kg and up to 4000 MICROLITERS/kg have demonstrated
efficacy in animal models when delivered as a single bolus to the
jugular vein, either before or after carotid artery ligation.
However, according to alternative embodiments, dosing of about 200
to 10,000 microliters per kg, about 600 to 2,000 microliters per
kg, and about 1,000 to 1,200 microliters per kg may be delivered as
a single bolus as a therapeutic dose.
[0068] Turning to FIG. 19, according to another embodiment of the
present disclosure, PC12 cells were cultured in DMEM containing 10%
FBS for 3 days, then starved in BME medium without FBS for 24
hours. Various percentages of the medium was exchanged for an
equivalent volume of ASC conditioned media, as indicated on the
figure above. The cells were cultured for 8 days in these media,
which were replaced with freshly made equivalent media every second
day. The number of cells that formed a neuronal phenotypes were
quantitated using a phase-contrast microscope. The results are
expressed as the mean percentage of neurite-bearing cells.+-.sd,
indicating that the ASC conditioned media induces differentiation
to neurite-bearing cells.
II. Experimental Support of Efficacy Across Neuronal Degenerative
Models
[0069] It will be appreciated that treatment of neural tissue
according to certain embodiments disclosed in the present
application were evaluated against several in vitro neuronal
degenerative models to demonstrate the effectiveness of the
treatment and composition with regard to multiple and varying types
of damage that can induce neuronal death. These models are well
established tools for the study of the CNS and peripheral nervous
system related diseases, disorders and injuries. (Ni, 1997; Du,
1997a; Du, 1997b; Dodel, 1998; Dodel, 1999; Du, 2001; Lin, 2001;
Lin, 2003). The use of these various models, which produce
reasonable similes of prevalent human diseases, are particularly
powerful tools for the study of the broad range of effectiveness of
the ASC Conditioned Media because each model is defined by distinct
mechanisms involving varied pathways of degeneration. Furthermore,
it is well known to those knowledgeable in the art that
interference of the distinct mechanisms involved in cellular
degeneration is limited to discrete factors and, furthermore, that
individual factors that act on one mechanism have no effect on
others. Individual trophic factors can modify only discrete
degenerative pathways or mechanisms. Therefore, a specific factor
would be expected to protect neurons from degenerative mechanisms
involved in specific neuronal death models, but would not provide
any protection in models involving damaging agents that induce
unrelated mechanism. Therefore, a single factor is unable to
protect neurons from all neuronal death mechanisms. However, a
mixture of factors, as is present in ASC Conditioned Media, would
provide the full complement of factors, acting individually or in
combination, necessary to block all degenerative mechanisms causing
cell death. In FIG. 16 it is shown that ASC Conditioned Media
possesses a factor (BDNF) that protects neurons from
glutamate-induced death. Neutralization of this factor greatly, but
not totally, reduces cell death in this model. Conversely, tests in
an in the mechanistically distinct LK/HK death model demonstrated
by neutralization of BDNF that this factor is not important for
modifying mechanisms leading to neuron death in this model.
Therefore, BDNF as an individual factor, as example, would not
protect neurons from all mechanisms causing neuron death.
[0070] Detailed descriptions of the major neuronal death pathways,
the involvement of each pathway in the models used in this study
and the relevance of each model to human disorders is described in
greater detail as follows below.
[0071] A. Mitogen-Activiated Protein Kinase
[0072] Mitogen-activated protein (MAP) kinases are widely expressed
serine-threonine kinases that mediate important regulatory signals
in cells. Three major groups of MAP kinases exist: the
extracellular signal-regulated (ERK) kinase family, the c-Jun
NH.sub.2-terminal kinase (JNK) family, and the p38 MAP kinase (p38)
family. The members of the different MAP kinase groups participate
in the generation of various cellular responses including gene
transcription, induction of cell death, maintenance of cell
survival, malignant transformation, and regulation of cell-cycle
progression (Widmann, 1999). The ERK-pathway is activated in
response to several cytokines and growth factors and primarily
mediates mitogenic and anti-apoptotic signals (Chang, 2001). There
are three isoforms of JNK. At least one of the JNK.sub.1-3 MAP
kinases is activated in response to stress and growth factors and
similarly mediates signals that regulate apoptosis, cytokine
production (inflammation), and cell-cycle progression (Davis,
2000). JNK signaling has been shown to be involved in transient
hypoxia-induced apoptosis in developing brain neurons (Chihab,
1998) and targeted deletion of JNK.sub.3 protected adult mice from
brain injury after cerebral ischemia-hypoxia (Kuan, 2003).
Additionally, blockade of JNK rescues neurons against potassium
deprivation-induced CGN death (Xifro, 2005) and glutamate-induced
neurotoxicity (Munemasa, 2005). p38 MAP kinase was discovered as a
major protein activated by LPS in macrophages and has been
characterized as the target for anti-inflammatory drugs that
inhibit IL-1 and TNF biosynthesis in monocytes (Lee, 1994; Han,
1994). Members of the p38 MAP kinase group are primarily activated
by stress stimuli, but also during engagement of various cytokine
receptors by their ligands (Lee, 1994; Lu, 1999; Rincon, 1998;
Wysk, 1999). The function of p38 kinases is required for the
generation of various activities including regulation of apoptosis
and cell cycle arrest, induction of cell differentiation, as well
as cytokine production and inflammation (Dong, 2002). p38 MAP
kinase also phosphorylates and/or modulates the activity of a
number of transcriptional factors involved in cytokine responses
including STAT1, IFN.sub..gamma. regulatory factor-1, and
NF-.kappa.B (Beyaert, 1996; Vanden Berghe, 1998). Recently, it has
been reported that inhibition of p38 MAP kinase significantly
inhibits NO-(Ghatan, 2000; Oh-hashi, 1999; Du, 2001),
glutamate-(Kawasaki, 1997) and possibly hypoxia-ischemia-induced
neuronal death (Hee, 2002).
[0073] Many of the genes responsible for apoptotic cell death,
including those underlying neuronal apoptosis, have now been
identified and named as caspases (Du, 1997a). Apoptotic cell death
is often mediated by a caspase cascade. Although many stimuli
exist, the final phases of apoptosis are executed by a few common
effector caspases. Mitochondria appear to provide a link between
the initiator caspases and the downstream effector caspases. In
non-neuronal cells, mitochondria have been shown to accelerate
activation of caspases by releasing pro-apoptotic molecules, such
as cytochrome c (Yang, 1997). MAP kinases such as JNK and p38 have
been reported to regulate caspase 3-mediated cell death (Harada,
1999; Cheng, 2001). However, it has also been reported that c-Jun
and p38 MAP kinases do not induce neuronal death through the
caspase-3 pathway (Sang, 2002; Roth, 2000). We have identified the
involvement of caspase 3 in cytochrome c-mediated, glutamate-(Du,
1997a), MPP-(Du, 1997b), 6-hydroxdopamine-(Dodel, 1999), and
potassium-deprivation-induced neurotoxicity (Ni, 1997).
Additionally, it has been suggested that caspase 3 plays a role in
the rat H-I model (Turmel, 2001). Further, it has been documented
that cytochrome c and caspase 3 have more important function in the
premature brain than the mature brain (Xu, 2004).
[0074] B. LK Induced CGN Apoptosis Model of Neuronal Cell Death
[0075] To induce apoptosis under the LK CGN model disclosed by Ni
in 1997, CGN maintained in BME with 25 mM potassium are switched to
regular BME (5 mM potassium) without serum and CGN (>50%), which
induces apoptosis within 24 h (Ni, 1997). This model was one of the
first to be established, and is still widely used in studies of
neuronal apoptosis in the primary cerebellar granule neuron (CGN)
culture system, although its precise relevance to the disease
remains unclear (D'Mello, 1993; Dudek, 1997, Ni, 1997).
[0076] In the developing rodent cerebellum, granule neurons undergo
developmentally regulated apoptosis peaking at the end of the first
week of postnatal life (Wood, 1993). Granule neurons cultured from
rats or mice around this time of development undergo cell death in
culture unless they are provided with extrinsic survival factors.
Maximal survival is produced by the combination of growth factors
typically provided by serum together with neuronal activity which
is induced by high extracellular concentrations of potassium
chloride that depolarize the membrane and induce activation of
voltage-sensitive calcium channels (D'Mello, 1997; Padmanabhan,
1999; Miller, 1996; Catterall, 2000). The signaling mechanisms by
which growth factors and neuronal activity promote the survival of
CGN are beginning to be characterized. Protein kinase cascades
figure prominently in the control of neuronal survival. The
ERK1/2-Rsk, phosphatidylinositol 3-kinase-Akt, and ERKS protein
kinase signaling pathways play critical roles in mediating the
survival of CGN upon exposure to the neurotrophin brain-derived
neurotrophic factor (Bonni, 1999; Shalizi, 2003). The
phosphatidylinositol 3-kinase-Akt signaling pathway plays a central
role in mediating insulin-like growth factor 1-mediated neuronal
survival (Brunet, 2001). Removal of survival factors promotes
neuronal apoptosis in part because of inactivity of pro-survival
protein kinases. However, deprivation of survival factors also
leads to stimulation of other protein kinases that impart an
apoptotic signal in neurons. These protein kinases include JNK,
p38, Cdc2, and GSK3 (Harada, 1999, Estus, 1994; Xia, 1995; Watson,
1998; Yang, 1997; Donovan, 2002; Konishi, 2002; Konishi, 2003;
Mora, 2001).
[0077] C. Glutamate Induced Model of Neuronal Cell Death
[0078] According to testing protocol for the glutamate induced
neuronal death model, neuronal apoptosis or necrosis is induced in
CGN with 30-100 .mu.M glutamate or cortical neurons (CN) with 100
.mu.M of NMDA. Glutamate is an excitatory neurotransmitter used
throughout the central nervous system and is associated with
various brain functions, such as synaptic plasticity, learning, and
long-term potentiation (Collingridge, 1989). Its physiological and
pathological effects in the CNS are mediated mainly via two types
of ionotropic glutamate receptors, the NMDA receptor and the
non-NMDA receptor. When present in excessive concentrations
glutamate has the potential to induce serious damage and even death
of neurons (Lucas, 1957), with N-methyl-D-aspartate (NMDA)
receptors located on neuronal cell bodies playing a major role in
this excitotoxicity (Rothman, 1987). NMDA receptor activation
allows an influx of calcium through both glutamate-activated
cationic channels (NMDA) and voltage-gated Ca.sup.2+ channels
activated by a prolonged depolarization (Choi, 1987; Coulter, 1992;
Olney, 1971). Although increases in intracellular calcium
concentrations are a necessary component of many normal signal
transduction pathways, excessive and prolonged rises of Ca.sup.2+
can lead to mitochondrial membrane dysfunction and cell death
(Farber, 1981; Sombati, 1991), induced in part by
Ca.sup.2+-mediated excitotoxicity (Wahlestedt, 1993) and/or failure
to regulate cell volume (Pasantes-Morales, 2000). Cell death
associated with glutamate neurotoxicity has been suggested to
contribute to the devastating effects of a number of serious
medical conditions including stroke, persistent seizures of status
epilepticus, and neurodegenerative disorders such as Alzheimer's
disease, amyotrophic lateral sclerosis, multiple sclerosis, spinal
cord injury and Huntington's disease (Choi, 1988; Choi, 1990;
Kandel, 1991).
[0079] It has been reported that reactive oxygen species (ROS) are
generated by activation of the glutamate receptor (Campisi, 2004).
Additionally, MAP kinases including JNK and p38 are also implicated
in glutamate-induced neuronal apoptosis (Xia, 1995; Chen, 2003).
Furthermore, caspase 3 activation appears to play an important role
in glutamate neurotoxicity (Du, 1997).
[0080] D. Hydrogen Peroxide Induced Model of Neuronal Cell
Death
[0081] According to the hydrogen peroxide induced death model, we
treated CGN with 10 .mu.M of H.sub.2O.sub.2 to induced neuronal
death (Lin, 2003). It has been suggested that hydrogen peroxide
leads to apoptotic neuronal death by involving pro-apoptotic
molecules (Wei, 2000), like initiator caspases (See, 2001).
Superoxide anions seem to be responsible for the apoptotic cell
death of trophic factor-deprived sympathetic neurons (Greenlund,
1995a; b), glutamate-treated cerebellar neurons (Ishikawa, 1999;
Satoh, 1998; and Patel, 1996), and hippocampal neurons incubated
with xanthine oxidase (Guo, 1999; Ishikawa, 1999). Singlet oxygen
has also been involved in apoptotic death in normeuronal cells
mediated by Bid and some members of the MAPK family (Zhuang, 1998).
In addition, singlet oxygen has been related to the alterations in
the mitochondrial permeability transition pore that occur in
several apoptotic death models (Salet, 1997; Moreno, 2001). ROS
contributes to the production of peroxynitrites and could also have
relevance in induction of apoptotic cell death (Virag, 1998).
[0082] E. Nitric Oxide Induced Model of Neuronal Cell Death
[0083] Treatment of CGN with 50 .mu.M of sodium nitroprusside (SNP,
a NO donor) induces neuronal death (Lin, 2001). Nitric oxide (NO)
generated from neuronal nitric oxide synthase (nNOS) and
inflammatory inducible isoform of nitric oxide synthase (iNOS)
inhibits the mitochondrial respiratory chain in vitro (Clementi,
1998), stimulates neurotransmitter release from synaptosomes
(Meffert.TM., 1994) and can cause autocrine excitotoxicity in
neuronal cultures (Leist, 1997). NO plays a critical role in
neurodegenerative diseases and cerebral ischemia. It has been
suggested that excessive production of NO causes these diseases by
destroying neurons. The mechanisms proposed for NO-mediated
neurotoxicity include inactivation of the mitochondrial respiratory
chain (Heales, 1994), S-nitrosylation of glyceraldehyde-3-phosphate
dehydrogenase (McDonald and J. Moss, 1993), inhibition of
cis-aconitase (Drapier, 1993), activation of poly (ADP-ribose)
synthase, and DNA damage (Zhang, 1994), most of which can be
mediated by the formation of nitrosocompounds by cellular
components. Additionally, p38 MAP kinase and cGMP-dependent protein
kinase (PKG) have been implicated in NO-induced neuronal apoptosis
(Ghatan, 2000; Lin, 2001; Bonthius, 2004).
[0084] F. 6-Hydroxy Dopamine Neuronal Model of Neuronal Cell
Death
[0085] Treatment of CGN or dopaminergic neurons (DA) with
6-hydroxydopamine ("6-OHDA") induces neuronal death (Dodel, 1999).
6-OHDA is a neurotoxin that is specific for
catecholamine/dopaminergic neurons (DN) in both the central and
peripheral nervous systems. This neurotoxin has been widely used
for the Parkinson disease (PD) research. It has been hypothesized
that 6-OHDA induces neuronal death possibly via uncoupling
mitochondrial oxidative phosphorylation resulting in energy
deprivation (Glinka, 1996). Alternatively, 6-OHDA-induced
neurotoxicity has been associated with its rapid auto-oxidation at
neutral pH, thus producing hydrogen peroxide, hydroxyl and
superoxide radicals (Kumar, 1995; Tiffany-Castiglinoi, 1982).
Quinones formed during the auto-oxidation of 6-OHDA may undergo
covalent binding with nucleophilic groups of macromolecules such as
--SH, --NH.sub.2, --OH, possibly further enhancing 6-OHDA-induced
neurotoxicity (Izumi, 2005). Furthermore, peroxynitrite
(ONOO.sup.-), which is a potent oxidant formed during the nearly
instantaneous reaction of nitric oxide with superoxide anion, has
also been found to be involved in 6-OHDA-induced neurochemical
effects (Ferger, 2001) and neurotoxicity (Mihm, 2001).
Peroxynitrite-mediated protein nitration has been well documented
in neurodegenerative disorders including Parkinson's disease
(Beckman, 1993; Good, 1996, 1998). We used CGN since CGN undergo
cell death as do dopaminergic neurons when exposed to 6-OHDA and
MPP.sup.+ (Dodel, 1999; Du, 1997a). Importantly, neuronal death
pathways have been better characterized in CGN since this system
provides a pure neuronal culture (Dodel, 1999).
[0086] G. MPP+ Induced Neuronal Model of Neuronal Cell Death
[0087] Treatment of CGN or dopaminergic neurons (DA) with MPP+
induces neuronal death (Du, 2001). MPTP/MPP.sup.+-induced
neurodegeneration of DAN and CGN is widely used to investigate and
characterize the pathogenesis of PD (Du 2001). MPP.sup.+ is
incorporated into cells via the dopamine transporters and the main
targets of MPP.sup.+ are the mitochondria, where it inhibits
Complex I in the respiratory chain and abolishes oxidative
phosphorylation (Tipton, 1993). Although the cerebellum has not
been extensively studied as a target for MPP.sup.+ neurotoxicity,
CGCs are quite sensitive to the toxic effects of MPP.sup.+ in vitro
(Du, 1997a; Gonzalez-Polo, 2003). The neurotoxic action of this
compound is known that MPP.sup.+ binds to complex-I of the
mitochondrial respiratory chain, causing the inhibition of
NAD-linked mitochondrial respiration (Javitch, 1985), the increase
in the generation of reactive oxygen species (Akaneya, 1995) and
caspase-3 activation (Du, 1997a). It has been also suggested the
regulatory effects of MPP.sup.+ on the N-methyl-D-aspartate
(NMDA)-receptor, inducing the Ca.sup.2+ entry into the cell
(Robinson and Coyle, 1987).
[0088] H. Hypoxia-Ischemia Neuronal Model of Neuronal Cell
Death
[0089] Hypoxic-ischemic (H-I) encephalopathy during the prenatal
and perinatal period is a major cause of damage to the fetal and
neonatal brain resulting in considerable morbidity and mortality
(Wei, 2004). However, currently, there is no effective treatment to
prevent the consequences of neonatal H-I in humans. Both rat and
mouse in vitro and in vivo models of neonatal H-I have been
established for mechanistic study. Hypoxic-ischemic insults can
trigger both apoptosis (delayed programmed cell death) and
necrosis. It has been reported that young neurons die of necrosis
and delayed apoptosis (Northington, 2001), whereas adult neurons
usually die of necrosis only (Walton, 1999). This difference is
mainly due to the upregulation of NMDA receptors and increased
caspase-3 activity in the young brain and these two factors make
young neurons particularly vulnerable to H-I injury (Johnston,
2002). Mitochondria appear to play an essential role in determining
the fate of cells subjected to hypoxia-ischemia (Gilland, 1998).
Disrupted mitochondrial function during H-I can lead to cytochrome
c protein release and trigger an activation of caspase 3/other
caspase-related apoptotic pathways (Cheng, 1998). Additionally,
Calpain and neuroinflammation may also be involved in H-I-induced
neuronal injury (Arvin, 2002, Wei, 2004). The prominence of both
apoptosis and necrosis in neurodegeneration after H-I in the
immature brain suggests that it will be important to better
understand the roles and relationships of these processes to
develop effective neuroprotective strategies.
[0090] I. Oxygen and Glucose Deprivation Neuronal Death Model
[0091] The in vitro oxygen and glucose deprivation (OGD) model
highly correlates to mechanisms of action in the in vivo H-I model.
We culture cortical (CN) or hippocampal (HN) neurons from 1-d pups
and after 7-d subject neurons to two hours of hypoxia in media
without glucose (see method for details). This model can be used
for mechanistic study of in vivo hypoxia-ischemia-induced neuronal
injury.
[0092] In summary, the table below lists some of the major neuronal
death pathways that are involved in the above-mentioned models.
TABLE-US-00001 TABLE 1 Models LK Glu H.sub.2O.sub.2 6OHDA MPP NO
OGD Cell type CGN CGN, CN CGN CGN, DN CGN, DN CGN CN, HN Necrosis
(N) or A N and A N and A N and A N and A N and A N and A apoptosis
(A) Caspase 3 weak strong weak weak strong weak modest JNK modest
modest weak weak modest weak modest p38 modest modest weak weak
weak strong modest Transcription/ strong weak weak weak weak weak
weak translation blocker Antioxidant weak* weak* Strong Some Some
Some modest Strong Strong Strong for some Physiology relevance
Apoptosis Ischemia All neuro- PD PD PD and Hypoxia model and others
degenerative inflammation- and ischemia disorders related neuronal
death *Some antioxidants may have neuroprotective functions through
non-antioxidant functions.
[0093] J. Assessment of Neuroprotective Effects Using
Neurodegenerative Models.
[0094] The in vitro and in vivo neuronal death models were used to
quantify the neuroprotective effect exerted by ASCs conditioned
media. These methods were further used to show the efficacy of
using various fractions and component of ASC conditioned media to
produce significant neuroprotective effects on different neuronal
death pathways. These techniques can also be used in vivo neonatal
H-I model to examine whether the conditioned medium or factors
identified therein can be systemically delivered to exert
neuroprotection in vivo.
III. Results of Testing ASC-CM Against Use of Neurodegenerative
Models
[0095] When the following neurodegenerative models were used by
incorporating ASC-CM into in vitro cultures, or according to
protocol set forth herein, the following results were noted.
[0096] A. ASC Conditioned Media Protects CGN Against
Glutamate-Induced Neuronal Death.
[0097] Glutamate induces both neuronal necrosis and apoptosis and
this in vitro model has been widely used for research of stroke,
Parkinson's disease, and Alzheimer's disease (Du, 1997). In order
to understand the physiological relevance of ASC Conditioned Media,
in the glutamate model was used to test neural cells cultured on
media containing ASC Conditioned Media. As shown in FIG. 6, the
conditioned medium from ASCs significantly protected neurons
against glutamate neurotoxicity. Furthermore, FIG. 7 demonstrates
that ASCs which had not been previously cultured ("fresh") produce
an ASC conditioned Media that possesses a higher potency in
neuroprotection than ASCs that are not fresh. Further, FIG. 8
indicates that ASC Conditioned Media that still retains the
protective/regenerative characteristics fractionate by size
exclusion chromatography at apparent molecular weights in excess of
10 kDa.
[0098] B. Human ASC-Conditioned Media Protects CGN Against
Glutamate-Induced Rat Neuronal Death.
[0099] Human ASC Conditioned Media was tested in a rat glutamate
toxicity model. The results indicate that human ASC Conditioned
Media, like that of rat, significantly attenuated glutamate
neurotoxicity in rat CGN, suggesting that ASC Conditioned Media
induce activity in rat cells. Thus, the rodent model can be useful
for assaying the neuroprotective properties of human ASCs.
[0100] C. ASC Conditioned Media Protects CGN Against
H.sub.2O.sub.2-Induced Neuronal Death.
[0101] The H.sub.2O.sub.2-induced neuronal death model shows the
role of free radicals in neurodegeneration. Free radicals have been
implicated in almost all types of neurodegenerative processes. Test
results shown in FIG. 12 demonstrate that ASC Conditioned Media
exerted potent anti-oxidant activity, thereby protecting CGN from
oxidative damage and death.
[0102] D. ASC Conditioned Media Protects Against OGD-Induced
Cortical Neuronal Death.
[0103] The oxygen and glucose deprivation (OGD) model highly
correlates to mechanisms in action in the in vivo H-I model. The
protective effect of ASC Conditioned Media was tested when added to
primary mouse cortical neurons from 1-d old pups. The cultured
neurons were placed in Hanks buffer without glucose and incubated
for 2 hours in a hypoxic chamber (Form a Scientific) that was
preset at 37.degree. C. and 1% O.sub.2. Neurons were then switched
back to serum-free DMEM medium in the presence or absence of ASC
Conditioned Media. 24 hours later, neurons were assayed by an LDH
kit. As a control, neurons without OGD treatments were also
switched into serum-free DMEM media in the presence or absence of
ASC Conditioned Media to eliminate LDH effects from ASC Conditioned
Media. Test results shown in FIG. 13 indicate that ASC Conditioned
Media markedly protects neurons against OGD-induced neuronal
injury.
[0104] E. 250.times. Enriched ASC Conditioned Media Protects
Neurons Against H-I-Induced Hippocampal Neuronal Death In Vivo.
[0105] To investigate ASC Conditioned Media function in H-I-induced
neuronal death in vivo, 7-d old Sprague Dawley rat pups were
anesthetized with 2.5% halothane and the left carotid artery was
permanently ligated. Hypoxic exposure was then achieved by placing
pups in a 2.0-L airtight plastic chamber submerged in a
37.0.degree. C. water bath and flushed for 2 h with a humidified
mixture of 7% oxygen and 93% nitrogen. Pups were then returned to
their dams until sacrifice. Pups (2 per group) received Intravenous
(i.v.) injections of 10 .mu.l of 250-fold concentrated rat ASC
Conditioned Media 24 h after the hypoxic insult.
[0106] The time period between H-I induction and ASC Conditioned
Media injection was chosen because maximal disruption of the
blood-brain barrier occurs at this time, allowing maximal
penetration of large polypeptides into brain tissues (Ikeda, 1999;
McLean 2004. Seven days following H-I injury, the brains were
histologically analyzed to quantify the amount of damage to the
hippocampus. In the hippocampus, H-I injury resulted in
approximately 27% tissue loss when mice were exposed to hypoxia for
2 hours, as compared to non-injured controls. Conversely, FIG. 12
shows that mice treated with ASC Conditioned Media showed almost
completely blocked brain damage.
[0107] As discussed above, ASC Conditioned Media effectively blocks
neuronal death in models that involve different molecular
mechanisms. These mechanisms include at least one of the following
three pathways: JNK, p38, and caspase 3. These three pathways have
been widely investigated and it is known that in addition to
interacting each other, these pathways may also induce neuronal
death independently (see Table I). Using these models, it is
possible to determine if ASC-conditioned Media (ACASC Conditioned
Media) inhibits injury stimuli-induced activation by
phosphorylation of JNK and p38 and cleavage of caspase 3 in those
models where they are actively involved (Table 1). According to our
embodiment, a ASC Conditioned Media prepared as described protects
neurons from neuronal death in these models via inhibition of ENK,
JNK, p38, and/or caspase 3 activation.
[0108] The adipose tissue is minced (mouse and rat) then digested
in Collagenase Type I solution (Worthington Biochemical, Lakewood,
N.J.) under gentle agitation for 1 hour at 37.degree. C., filtered
with 500 .mu.m and 250 .mu.m Nitex filters, and centrifuged at 200
g for 5 minutes to separate the stromal cell fraction (pellet) from
adipocytes. The ASC fraction is treated with red blood cell lysis
buffer for 5 min at 37.degree. C., then centrifuged at 300 g for 5
minutes. The supernatant is discarded and the cell pellet
resuspended in the appropriate medium.
[0109] F. ASC Conditioned Media Protects Against OGD-Induced
Cortical Neuronal Death.
[0110] ASC-CM protects neurons against 6-hydroxydopamine
(6-OHDA)-mediated death, as shown in FIG. 18. The ASC Conditioned
Medium (ASC-CM) was collected and subsequently added to the
cultured rat cerebellar granule neurons (CGN). Neuronal viability
was quantified by either counting fluorescein positive neurons or
staining living neurons with MTT. Since neurotoxicity induced by
6-OHDA was believed to be due, at least in part, to the production
of reactive oxygen species (ROS). Also investigated were the levels
of free radical generation in our model by using dihydroethidium
(DHE) and dihydrorhodamine 123 (DHR). As shown in FIG. 18, exposure
of CGN to 50 mM 6-OHDA resulted in significant increases in free
radical production and CGN neuronal death.
[0111] G. ASC-CM Preserves the Cognitive Function of Rats Following
Hypoxia-Ischemia Injury.
[0112] The ability of ASC-CM to provide long-term protection
following hypoxia-Ischemia (HI) injury was determined as follows.
HI injury was induced in 7 day old rat pups as described above.
ASC-CM was administered at the time of surgery (pre-treatment) or
24 hours after HI injury (post treatment). Controls were uninjured
rats (positive control) of the same age and rats receiving an
equivalent volume of carrier (negative control). After 7 weeks the
cognitive function of all rats was determined using the Morris
Water Maze test.
[0113] The test system consisted of a swimming pool containing a
number of visual cues to facilitate orientation, including counters
and decals. A 168 cm diameter, 41 cm high tank was filled to a
depth of 30 cm with 15.degree. C. water. A round transparent
plastic platform, 11 cm in diameter, was placed in the pool so that
the top of the platform was located 1 cm below the surface of the
water, where it was not visible to a viewer on the surface of the
water. For the visible platform test, a flag was placed on the
platform. After performing the visible platform test, the flag was
removed, making the platform not visible from the surface of the
water (invisible test). Animals were individually placed at the
same location in the water to begin the test. The time taken for
the rats to reach the platform by swimming was recorded. Each
animal was tested 3 times with 15 second intervals between repeats.
Data are presented as mean.+-.SEM. The results were compared using
a paired Student's t-Test
[0114] As shown on FIG. 17, for the visible platform test the time
taken by the rats to first swim to the platform and then crawl out
of the water onto the platform was much shorter for both
ASC-pretreatment (n=3) and ASC-posttreatment (n=4) groups than for
the control BME-treatment group (n=5) (**P<0.01). Similarly,
ASC-CM treated rats performed better than BME control-treated rats
in the invisible platform test (*P<0.05) (FIG. 2). These results
demonstrate that ASC-CM treated rats have a higher level of
cognitive function than control treated animals; thus, providing
further evidence that ASC-CM provides protection against
neurodegeneration.
IV. Methods
[0115] A. Preparation of Mouse and Rat CGN Neuronal Cultures and
Analysis Method
[0116] CGN is prepared from 8-day-old rat or mouse pups as
previously described (Du, 1997 and 2001). Preliminary data showed
that mouse CGN behaves similarly to rat CGN. Briefly, freshly
dissected cerebella is dissociated and the cells seeded at a
density of 1.2 to 1.5.times.10.sup.6 cells/ml on poly-L-lysine
coated dishes in basal medium Eagle (Invirogen) supplemented with
10% FBS (Invirogen), 25 mM KCl, and gentamicin (0.1 mg/ml,
Invitrogen). Cytosine arabinoside (10 .mu.M, Sigma) is added to the
culture medium 24 h after initial plating. All experiments utilize
neurons after 7-8 days in vitro (DIV). The LK, glutamate, H2O2,
MPP+, 60HDA treatments follow methods that were previously
described (Ni, 1997; Du, 1997a; Lin, 2003; Du, 1997b; Dodel, 1999).
Viable neurons are quantified by counting fluorescein (green)
positive cells which result from the de-esterification of
fluorescein diacetate (FDA, Sigma) by living cells. Briefly,
cultures are incubated with FDA (10 .mu.g/ml) for 5 min, examined
and photographed using UV light microscopy and the number of
neurons from representative low power fields are counted as
previously described (Du, 1997). Propidium iodide (PI, Sigma),
which interacts with nuclear DNA producing a red fluorescence, is
used to identify dead neurons. For PI staining, cultures are
incubated with PI (5 .mu.g/ml), examined and photographed using UV
light microscopy as previously described (Du, 1997a).
[0117] B. Cultured Mesencephalic Neurons
[0118] Primary cultures of rostral mesencephalic tegmentum (RMT)
dissected from E15 rat or E12 mouse embryos (Harlan) are performed
using a modified method as previously described (Dodel, 1998).
Preliminary studies show that mouse MDN behaves similarly to rat
MDN. Briefly, RMT is dissociated using trypsin and DNase (Sigma)
and the cells are be suspended in Dulbeccos Modified Eagle Medium
(Invitrogen) supplemented with Ham F12 nutrient mixture (1:1;
Invitrogen), glucose, 1% penicillin-streptomycin (Invitrogen) and
10% fetal bovine serum (Invitrogen). The cells are plated onto
poly-L-lysine (10 .mu.g/ml; Sigma) precoated 10 mm coverslips in
24-well plates at a density of 10.sup.5 cells/cm.sup.2 and
incubated for 72 hr. Following 24 h the medium is supplemented with
10 .mu.M cytosine arabinoside (Sigma) to inhibit glial cell
proliferation. Neuronal cultures are used for experiments 7 days
after preparation. Dopaminergic (DA) neurons in primary cultures
are visualized by TH-immunohistochemistry using a primary
monoclonal antibody against rat TH (Incstar) following by an
anti-mouse IgG Cy3 conjugate (Sigma) (Dodel, 1998), and the number
of TH-immunoreactive neurons is assessed using a Leitz inverted
microscope (.times.200). Values are usually expressed as a % of
control cultures for each experiment and the data are displayed as
the mean.+-.standard error of duplicate experiments, which are
repeated about four times. The cell counts are statistically
evaluated using analysis of variance.
[0119] Neurotoxicity is also examined by using methods of TUNEL
(APOPTAG, ONCOR) and LDH (Roche) following manufacturers'
instructions (Dodel, 1998).
[0120] C. Primary Neonatal Cortical Neuronal Culture
[0121] Cortices are collected from newborn rat or mice pups and
minced. An aliquot of ice-cold PBS is added into the minced
tissues, which are then centrifuged at 1000 rpm at 4.degree. C. and
the supernatants are discarded. An aliquot of 0.25% trypsin is
added and incubated at 37.degree. C. for 15 min to produce a single
cell suspension (shaken once every 5 min). The precipitates are
discarded and the supernatants are centrifuged again at 1000 rpm at
4.degree. C. for 5 min. The cell pellets are diluted to an
appropriate concentration with Neurobasal in 2% B27 (Invitrogen,
Carlsbad, Calif., U.S.A.) and plated into poly-d-lysine-coated
dishes (BD Biosciences, Franklin Lakes, N.J., U.S.A.). Usually, the
cells are used between 4-6 days after plating. Before each
treatment, cells are rinsed and then incubated in serum-free
Dulbecco's modified Eagle's medium (DMEM) with or without high
glucose (Invitrogen). All experiments are conducted under
serum-free conditions. To induce OGD, neurons are placed in a
hypoxic chamber (Form a Scientific) which is preset at 37.degree.
C. and 1% O.sub.2. Neurons are incubated with serum-free DMEM media
containing no glucose. Control neurons are incubated in the regular
incubation chamber (37.degree. C. and 21% O.sub.2) in DEME
containing high glucose. Four hours later, neurons are placed back
to regular CO.sub.2 incubator for another 20 h and then assayed
using a LDH kit. For NMDA toxicity study, the neuronal culture is
supplemented with 100 mM for 24 h and assayed using a LDH kit.
[0122] D. MTT Assay
[0123] Cell viability assays are performed in accordance with the
protocol provided by R & D Systems (Minneapolis, Minn.,
U.S.A.). Briefly, cortical neurons from newborn rats are cultured
in flat-bottomed, poly-d-lysine-coated, 96-well tissue culture
plates (BioCoat, BD Biosciences). After each treatment, 100 .mu.l
of media is removed for the LDH assay and MTT is added to the
cultures at 37.degree. C. for 2.5 h. DMSO is then added to the
cells. Cells are held for another 3 h at 37.degree. C. in the dark
since MTT is reduced by metabolically active cells into insoluble
purple formazan dye crystals that are soluble in the DMSO. The
absorption is read by a plate reader at 570 nm using a reference
wavelength of 650 nm.
[0124] E. LDH Assay
[0125] About 100 .mu.l of the culture media is monitored for the
release of lactate dehydrogenase (LDH) to measure cell death, using
a LDH kit from Roche, Indianapolis by following the manufacturer's
instructions (Du, 1998). Each experiment is performed in
triplicate; the data from a representative experiment carried out
three times with similar results. The data is expressed as the mean
OD.+-.SD.
[0126] F. TUNEL Assay
[0127] DNA strand breaks are detected using terminal
deoxynucleotidyl transferase-mediated biotinylated UTP nick
end-labeling (TUNEL) according to the manufacturer's procedure
(APOPTAG, ONCOR). Briefly, cultures are fixed for 30 min with 1%
paraformaldehyde and then washed with PBS. 200 .mu.l of
Equilibration buffer is added to each well, followed by addition of
120 .mu.l/well of working strength TdT cells are, then incubated
for 30 min at 37.degree. C. After adding 1 ml of working strength
stop/wash buffer twice, 100 .mu.l of working strength antibody
solution (anti-digoxigenin-fluorescein) is added the mixture is
held for 1 hr. The cells are visualized under phase-contrast
microscopy. Apoptotic cells are discriminated morphologically by
the presence of condensed, bright green nuclei in neurons.
[0128] G. Western Blot Analysis
[0129] Western blot analysis of ERK, JNK, p38, and active caspase
is performed as previously described (Wei, 2004, Wei, 2005).
Detection of caspase activity is also performed as described in
1997 (Du, 1997a,b). Neuronal extracts are prepared by lysing CGN at
1.5 and 3 h (for JNK and p38), 0, 3 and 6 h (ERK), and 20 h (for
caspase 3) following insult treatments.
[0130] H. Proteomic Profiling of Neuroprotective Factors in
Fractions from ASC-Conditioned Media
[0131] Neuroprotective factors secreted by ASCs, are characterized
using antibody arrays to identify specific factors present in
fractions of conditioned medium. This information is used to assess
the contribution of each factor to neuroprotective activity. An
antibody array is used for initial characterization over other
proteomics analyses because this method is sensitive (can detect pg
level) and can directly measure the protein in media. In contrast,
nucleic acid microarrays (SuperArray, Affymetrix, Agilent) can only
detect mRNA changes and may not provide accurate data on protein
production and secretion in to the media. Other methods for
detecting proteins are much more powerful and can be used. Our data
demonstrates that conditioned media from human ASCs potently
protects rat neurons, suggesting that cross-species analyses and
protection occurs.
[0132] I. Proteomic Profiling of Growth Factors/Cytokines Present
in Conditioned Media
[0133] Active fractions from human and mouse ASC antibody can be
identified using array membranes provided by RayBiotech. Detailed
methodology is described in the company protocol; it is similar to
Western blot protocols. In brief:
[0134] Step 1. Incubate the array membrane with 250-fold enriched
ASCs supernatants.
[0135] Step 2. Incubate the factor-bound membrane with a cocktail
of biotin-labeled antibodies.
[0136] Step 3. Incubate the array membrane with HRP-conjugated
streptavidin.
[0137] Step 4. chemoluminescent detection.
[0138] The presence of any proteins detected by the array analyses
can be confirmed in rat or murine (if not proved to be negative
through probing the mouse array) conditioned media using antibodies
or RT-PCR analyses, and the identified proteins can be manufactured
using cDNA or other methods.
[0139] Because of the larger array of human antibodies, and
conditions available it is more effective to probe for human
proteins than it is to probe for specific proteins in rats, mice
and other animals. However, mouse and rat ASCs may have unique
properties that are not conserved across species. We do not believe
that this is the case since our data demonstrate that human ASC
conditioned media is a potent protector of rat neurons. However,
the number of proteins detected by the arrays is still a fraction
of the total number of proteins present in the media. It is
possible that important factors in the conditioned media could go
undetected. Accordingly, it may be necessary to use methods other
than antibody detection to screen for factors in ACASC Conditioned
Media produced by rat or mice cells. One such method is to use
oligonucleotide array to identify components produced by ASCs.
Commercially available oligonucleotide arrays include SuperArray,
Affymetrix, or Agilent. The other arrays can be used to probe for
factors that do not react with antibody assay. This technique
provides information in the absence of antibodies and can be used
directly with mouse and rat cells. If necessary, these two
methodologies can be combined to overcome these inherit
deficiencies of each separate method.
[0140] All references, patients, patient applications and the like
cited herein and not otherwise specifically incorporated by
references in their entirety, are hereby incorporated by references
in their entirety as if each were separately incorporated by
reference in their entirety.
[0141] An abstract is included to aid in searching the contents of
the application it is not intended to be read as explaining,
summarizing or otherwise characterizing or limiting the disclosure
in any way.
[0142] The present disclosure contemplates modifications as would
occur to those skilled in the art. It is also contemplated that
processes embodied in the present disclosure can be altered,
duplicated, combined, or added to other processes as would occur to
those skilled in the art without departing from the spirit of the
present disclosure.
[0143] Further, any theory of operation, proof, or finding stated
herein is meant to further enhance understanding of the present
disclosure and is not intended to make the scope of the present
disclosure dependent upon such theory, proof, or finding.
[0144] While the disclosure has been illustrated and described in
detail in the drawings and foregoing description, the same is
considered to be illustrative and not restrictive in character, it
is understood that only the preferred embodiments have been shown
and described and that all changes and modifications that come
within the spirit of the disclosure are desired to be
protected.
* * * * *