U.S. patent application number 15/910218 was filed with the patent office on 2018-07-19 for isolation of human neural stem cells from amniotic fluid of patients with neural tube defects.
The applicant listed for this patent is FOOD INDUSTRY RESEARCH AND DEVELOPMENT INSTITUTE. Invention is credited to Yu-Jen CHANG, Lee-Feng HSU, Shiaw-Min HWANG.
Application Number | 20180200303 15/910218 |
Document ID | / |
Family ID | 56924406 |
Filed Date | 2018-07-19 |
United States Patent
Application |
20180200303 |
Kind Code |
A1 |
HWANG; Shiaw-Min ; et
al. |
July 19, 2018 |
ISOLATION OF HUMAN NEURAL STEM CELLS FROM AMNIOTIC FLUID OF
PATIENTS WITH NEURAL TUBE DEFECTS
Abstract
The present invention provides a method for isolating human
neural stem cells from amniotic fluid of a patient whose fetus has
been diagnosed to have a neural tube defect. Use of the isolated
human neural stem cells in the treatment of neurological disorders
is also provided.
Inventors: |
HWANG; Shiaw-Min; (HSINCHU
CITY, TW) ; CHANG; Yu-Jen; (HSINCHU CITY, TW)
; HSU; Lee-Feng; (HSINCHU CITY, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
FOOD INDUSTRY RESEARCH AND DEVELOPMENT INSTITUTE |
HSINCHU CITY |
|
TW |
|
|
Family ID: |
56924406 |
Appl. No.: |
15/910218 |
Filed: |
March 2, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14660554 |
Mar 17, 2015 |
9943549 |
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15910218 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 35/30 20130101;
G01N 33/5014 20130101; G01N 33/5058 20130101; A61P 25/28 20180101;
A61P 25/00 20180101; C12N 5/0623 20130101 |
International
Class: |
A61K 35/30 20150101
A61K035/30; G01N 33/50 20060101 G01N033/50; C12N 5/0797 20100101
C12N005/0797 |
Claims
1. A method for treating a neurological condition in a mammal in
need thereof, comprising administering an isolated human neural
stem cells or a pharmaceutical composition comprising the isolated
human neural stem cells to the mammal, wherein the isolated human
neural stem cells are obtained from a method consisting of: (a)
collecting the cells from amniotic fluid obtained from a pregnant
human subject whose fetus has been diagnosed to have a neural tube
defect; (b) incubating the cells with a culture medium; (c)
detecting expression levels of Nestin, Sox2, Musashi-1, and
ATP-binding cassette G2 (ABCG2) markers and activity of
Stage-Specific Embryonic Antigen-3 (SSEA-3), Stage-Specific
Embryonic Antigen-4 (SSEA-4), Tumor Rejection Antigen-1-60
(TRA-1-60), TRA-1-81 and telomerase in the cells; and (d) isolating
from the culture medium human neural stem cells that express
Nestin, Sox2, Musashi-1 and ABCG2 markers and exhibit telomerase
activity but do not express SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81
from the culture medium.
2. The method of claim 1, wherein the neurological condition is a
neurological disease with pathophysiological mechanisms involving
ischemia, a neurological disease with pathophysiological mechanisms
involving hypoxia, a neurodegenerative disease, or a disease of the
nervous system accompanied by neural death.
3. The method of claim 1, wherein the neural tube defect is
anencephaly or myelomeningocele.
4. The method of claim 3, wherein the neural tube defect is
anencephaly.
5. The method of claim 1, wherein the culture medium is a
serum-free cell culture medium that allows the neural stem cells to
proliferate.
6. A method for screening drug candidates comprising: contacting an
isolated human neural stem cells with a drug candidate; and
determining one or more cell conditions of the cells, if the
determined one or more cell conditions are better than the same
condition(s) of the cells without contacting the drug candidate, it
represents that the drug candidate has potential in the treatment
of neurological conditions, wherein the isolated human neural stem
cells are obtained from a method consisting of: (a) collecting the
cells from amniotic fluid obtained from a pregnant human subject
whose fetus has been diagnosed to have a neural tube defect; (b)
incubating the cells with a culture medium; (c) detecting
expression levels of Nestin, Sox2, Musashi-1, and ATP-binding
cassette G2 (ABCG2) markers and activity of Stage-Specific
Embryonic Antigen-3 (SSEA-3), Stage-Specific Embryonic Antigen-4
(SSEA-4), Tumor Rejection Antigen-1-60 (TRA-1-60), TRA-1-81 and
telomerase in the cells; and (d) isolating from the culture medium
human neural stem cells that express Nestin, Sox2, Musashi-1 and
ABCG2 markers and exhibit telomerase activity but do not express
SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 from the culture medium.
7. The method of claim 6, wherein the neural tube defect is
anencephaly or myelomeningocele.
8. The method of claim 7, wherein the neural tube defect is
anencephaly.
9. The method of claim 6, wherein the culture medium is a
serum-free cell culture medium that allows the neural stem cells to
proliferate.
10. A method for testing cytotoxicity of a drug candidate
comprising: contacting an isolated human neural stem cells with the
drug candidate; and determining one or more cell conditions of the
cells, if the determined one or more cell conditions are poorer
than the same condition(s) of the cells without contacting the drug
candidate, it represents that the drug candidate may have
cytotoxicity, wherein the isolated human neural stem cells are
obtained from a method consisting of: (a) collecting the cells from
amniotic fluid obtained from a pregnant human subject whose fetus
has been diagnosed to have a neural tube defect; (b) incubating the
cells with a culture medium; (c) detecting expression levels of
Nestin, Sox2, Musashi-1, and ATP-binding cassette G2 (ABCG2)
markers and activity of Stage-Specific Embryonic Antigen-3
(SSEA-3), Stage-Specific Embryonic Antigen-4 (SSEA-4), Tumor
Rejection Antigen-1-60 (TRA-1-60), TRA-1-81 and telomerase in the
cells; and (d) isolating from the culture medium human neural stem
cells that express Nestin, Sox2, Musashi-1 and ABCG2 markers and
exhibit telomerase activity but do not express SSEA-3, SSEA-4,
TRA-1-60 and TRA-1-81 from the culture medium.
11. The method of claim 10, wherein the neural tube defect is
anencephaly or myelomeningocele.
12. The method of claim 11, wherein the neural tube defect is
anencephaly.
13. The method of claim 10, wherein the culture medium is a
serum-free cell culture medium that allows the neural stem cells to
proliferate.
Description
FIELD OF THE APPLICATION
[0001] The present application relates to human neural stem cells.
More specifically, the present invention provides a method for
isolating human neural stem cells from amniotic fluid of a patient
whose fetus has been diagnosed to have a neural tube defect, and
the uses of the isolated human neural stem cells.
BACKGROUND
[0002] Neural stem cells (NSCs) found in the central nervous system
(CNS) have the capacity both to self-renew and to differentiate
into each of the major cell types in brain. Ever since they were
first described in mouse brain, NSCs have been the subject of
intensive investigation because of their potential therapeutic use
in treating neurodegenerative disorders [1, 2]. Specifically,
transplanting NSCs may induce cellular repair and recovery of
function after CNS injury or disease [3-6]. Previous studies have
demonstrated that NSCs grafted into the CNS not only form new
neurons but also express protective and trophic factors that are
released into the damaged area.
[0003] Previously identified sources of NSCs in the adult mammalian
CNS include the subgranular zone of the hippocampus and the
subventricular zone of the ventral forebrain [7]. Human NSCs are
typically obtained from aborted fetuses, post-mortem brains or
surgical specimens [7-9]. However, the variability in donor age,
storage, viability and potential contamination of these samples
make it difficult to use them in therapeutic applications [10].
Other barriers include limited availability, technical difficulty
in harvesting, and ethical concerns. Finally, the slow kinetics of
human NSCs growth in primary cultures imposes a severe limitation
on the ability to obtain enough quality cells for clinical
applications. Recently, some immortalized neural stem/progenitor
cell lines have been established [11, 12], which possess a
relatively higher capacity for proliferation than typical NSCs
while still retaining the ability to differentiate into different
neural cell types. However, the use of oncogenic genes and viral
infection in establishing these lines raises vital concerns over
risk in medical-oriented applications. Other groups have
established lines from pluripotent sources of stem cells such as
embryonic stem cells or induced pluripotent stem cells [13-15].
While these methods do introduce a new source of NSCs, the
possibility remains that undifferentiated cells will persist in
these populations and could consequently form teratomas [16].
Therefore, the ability to use pluripotent stem cell-derived NSCs
for therapeutic applications is limited by ethical issues, safety
concerns, and poor efficiency.
[0004] Neural tube defects (NTDs) are the most common defects when
a neural tube develops abnormally, and they affect approximately 1
in 1000 pregnancies [17]. The neural tube is formed during
embryonic development and eventually gives rise to the entire CNS.
When the neural tube does not close completely on either end, an
NTD occurs. In humans, the most common NTDs are anencephaly and
myelomeningocele. The former results from a failed closure of the
rostral end of the neural tube and is characterized by a total or
partial absence of the cranial vault and cerebral hemisphere, while
the latter is a defective closure of the caudal neural tube and the
vertebral column [18-20]. Anencephaly results in incomplete
formation of the brain and skull and is therefore lethal. Most
individuals with myelomeningocele have a multiple system handicap
and a limited lifespan. Either ultrasound technology or measurement
of maternal serum alpha-fetal protein levels can be used to detect
an NTD in utero [21]. Follow-up testing typically measures the
levels of alpha-fetal protein and acetylcholinesterase in the
amniotic fluid to confirm that an NTD is present [22].
[0005] Amniotic fluid (AF) is known to contain multiple cell types
that are derived from the developing fetus, and previous studies
have demonstrated that multipotent stem cells can be isolated from
this substance via amniocentesis. These AF-derived stem cells
(AFSCs) express some pluripotent markers and can differentiate into
cells of mesenchymal or neural lineages under inductive conditions
[23-25]. Although AFSCs exhibit neural potentiality both in vivo
and in vitro, they lack some typical properties of NSCs, such as
proper growth, morphology and the potential to form neurospheres.
To date, no group has been able to isolate NSCs directly from
normal amniotic fluid samples of any species. Recently, one group
reported that NSCs could be established from the amniotic fluid of
pregnant rats in which the fetus had an NTD [26].
[0006] Human neural stem cells (NSCs) are a particularly valuable
tool for the study of both nerve system development and the
function of adult neurogenesis. NSCs also have great therapeutic
potential in treating neurodegenerative disorders. However, current
sources of human NSCs are limited for technical reasons such as the
difficulty in isolating them and the time needed to expand the
population.
[0007] Therefore, there is still a need to develop a method to
obtain human NSCs which can be expanded for long periods without
losing their stem cell-specific properties.
SUMMARY OF THE INVENTION
[0008] One aspect of the invention relates to a method for
obtaining isolated human neural stem cells from amniotic fluid
obtained from a pregnant human subject whose fetus has been
diagnosed to have a neural tube defect, wherein the isolated human
neural stem cells express Nestin, Sox2, Musashi-1 and ATP-binding
cassette G2 (ABCG2) markers but do not express SSEA-3, SSEA-4,
TRA-1-60 and TRA-1-81.
[0009] Another aspect of the invention relates to isolated human
neural stem cells obtained from the method of the present
invention.
[0010] Another aspect of the invention relates to a pharmaceutical
composition comprising the isolated human neural stem cells
obtained from the method of the present invention and a
pharmaceutically acceptable carrier.
[0011] Another aspect of the invention relates to a method for
treating a neurological condition in a mammal in need thereof,
comprising administering the pharmaceutical composition of the
present invention to the mammal.
[0012] Another aspect of the invention relates to a method for
screening drug candidates, wherein the method comprises the steps
of contacting the isolated human neural stem cells obtained from
the method of the present invention with a drug candidate; and
determining one or more cell conditions of the cells; if the
determined one or more cell conditions are better than the same
condition(s) of the cells without contacting the drug candidate, it
represents that the drug candidate has potential in the treatment
of neurological conditions.
[0013] Another aspect of the invention relates a method for testing
the cytotoxicity of a drug candidate, wherein the method comprises
the steps of contacting the isolated human neural stem cells
obtained from the method of the present invention with the drug
candidate; and determining one or more cell conditions of the
cells; if the determined one or more cell conditions are poorer
than the same condition(s) of the cells without contacting the drug
candidate, it represents that the drug candidate may have
cytotoxicity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1A shows the culture properties of AF-NSCs. After
initial seeding of the amniotic fluid cells in NeuroCult NS-A
proliferation medium, neural-like cells began to attach to the
culture plate (left hand panel). These cells then proliferated and
rounded up to form suspended primary neurospheres (right hand
panel). Abbreviations: DIV: days in vitro.
[0015] FIG. 1B shows the culture properties of AF-NSCs. While the
neurospheres were dissociated into a single cell suspension for
plating, afterwards, they continued to divide and re-formed into
neurospheres. Mature AF-NSC-derived neurospheres contained a
typical micro-spike structure over the outer surface (right, black
arrows). Scale bar: 10 .mu.m. Abbreviations: DIV: days in
vitro.
[0016] FIG. 1C shows the culture properties of AF-NSCs. The
doubling time of AF-NSCs was affected by the seeding density. *:
p<0.05; **: p<0.01.
[0017] FIG. 1D shows the culture properties of AF-NSCs. AF-NSCs
cell counts from long-term in vitro cultures. Two different AF-NSC
lines were used for this analysis starting from passage 5, and the
total cell number was calculated cumulatively at each passage. The
two AF-NSCs lines, 100 and 106, were cultured for 18 and 23
passages, respectively. (Black square: AF-NSC line #100; black
circle: AF-NSC line #106)
[0018] FIG. 2A shows the expression of NSC-specific markers in
AF-NSCs. Confocal images of immunostained mature neurospheres (Day
14) revealed the expression of specific NSC markers (Nestin,
Musashi-1 and Sox2). Scale bar: 20 .mu.m.
[0019] FIG. 2B shows the expression of NSC-specific markers in
AF-NSCs. The expression of NSC-specific cell markers in AF-NSCs was
determined by flow cytometry at early and late passage numbers.
Dotted line: isotope antibody control; gray line: early passage
#(10-12); black line: late passage #(20-22). Abbreviations: SSEA:
stage-specific embryonic antigen; ABCG2: ATP-binding cassette
sub-family G2; HLA: human leukocyte antigen.
[0020] FIG. 2C shows the expression of NSC-specific markers in
AF-NSCs. qPCR was used to determine mRNA levels of NSC-specific
genes at early and late passages. White bar: early passage #10;
gray bar: late passage #20.
[0021] FIG. 2D shows the measurement of telomerase activity in
AF-NSCs. Telomerase activity was measured at different times in
culture (passage #6, 10 and 17). The triangle designates samples
that were treated by heat inactivation. Abbreviations: N: negative
control; P: positive control.
[0022] FIG. 3A shows in vitro differentiation of AF-NSCs. The
AF-NSCs were cultured in neural differentiation medium, and markers
of early and mature neurons were detected by immunocytochemistry at
2 days post-induction. Scale bar: 20 .mu.m. Abbreviations: DAPI:
4',6-diamidino-2-phenylindole.
[0023] FIG. 3B shows in vitro differentiation of AF-NSCs. The
AF-NSCs were cultured in neural differentiation medium, and markers
of early and mature neurons were detected by immunocytochemistry at
7 days post-induction. Scale bar: 20 .mu.m. Abbreviations: DAPI:
4',6-diamidino-2-phenylindole.
[0024] FIG. 3C shows the expression of neuronal specific genes
after in vitro differentiation of AF-NSCs. qPCR was used to measure
the expression levels of these neuronal markers. White bar:
undifferentiated cells; gray bar: differentiated cells at day 2;
black bar: differentiated cells at day 7. *: p<0.05; **:
p<0.01.
[0025] FIG. 4A shows directed differentiation of AF-NSCs into
astrocytes, oligodendrocytes and dopaminergic neurons. AF-NSCs were
cultured in specific differentiation medium and immunostained for
specific markers to confirm the presence of astrocytes (GFAP),
oligodendrocytes (O4) and dopaminergic neurons (TH and AADC). Scale
bar: 20 .mu.m. Abbreviations: GFAP: glial fibrillary acidic
protein; TH: tyrosine hydroxylase; AADC: aromatic L-amino acid
decarboxylase; and DAPI: 4',6-diamidino-2-phenylindole.
[0026] FIG. 4B shows the expression of specific markers of
differentiated AF-NSCs. qPCR was used to analyze gene expression of
astrocyte (GFAP), oligodendrocyte (CNP, MBP and O2) (left) and
dopaminergic neuron (AADC, Pax2, Lmx-1b and Nurr-1) (right)
specific markers. White bar: before differentiation; black bar:
after differentiation. *: p<0.05; **: p<0.01. Abbreviations:
GFAP: glial fibrillary acidic protein.
[0027] FIG. 5A shows engrafting AF-NSCs into MCAO ischemic rats has
therapeutic effects. After AF-NSCs were transplanted, ischemia rats
underwent a rotarod test. Black bar: healthy control; white bar:
sham control; gray bar: AF-NSC transplantation after MCAO. FIG. 5B
shows engrafting AF-NSCs into MCAO ischemic rats has therapeutic
effects. After AF-NSCs were transplanted, ischemia rats underwent a
grip strength test. Black bar: healthy control; white bar: sham
control; gray bar: AF-NSC transplantation after MCAO. FIG. 5C shows
engrafting AF-NSCs into MCAO ischemic rats has therapeutic effects.
Quantification of the hemispheric lesion area by TTC 4 weeks after
AF-NSC transplantation. FIG. 5D shows engrafting AF-NSCs into MCAO
ischemic rats has therapeutic effects. Top: Immunohistochemistry
revealed Nestin (green) expression in the injection region. Bottom:
Higher magnification of this region revealed that grafted AF-NSCs
co-express Nestin (green) and human Nuclei (red, arrows) in the
damaged area. Scale bar: 50 .mu.m.
[0028] FIG. 6 shows the karyotypes of the 4 AF-NSC lines that were
established in this study. All 4 of the AF-NSCs lines possess
normal karyotypes, including one 46XX and three 46XY.
DETAILED DESCRIPTION
[0029] The present invention can be understood more readily by
reference to the following detailed description of various
embodiments of the invention, the examples, and the tables with
their relevant descriptions. Unless otherwise defined, all terms
(including technical and scientific terms) used herein have the
same meaning as commonly understood by one of ordinary skill in the
art to which this invention belongs. It will be further understood
that terms such as those defined in commonly used dictionaries
should be interpreted consistently with their meaning in the
context of the relevant art and will not be interpreted in an
idealized or overly formal sense unless expressly so defined
herein. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments only
and is not intended to be limiting.
[0030] It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an" and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, unless otherwise required by context, singular terms shall
include the plural and plural terms shall include the singular.
[0031] Often, ranges are expressed herein as from "about" one
particular value and/or to "about" another particular value. When
such a range is expressed, an embodiment includes the range from
the one particular value and/or to the other particular value.
Similarly, when values are expressed as approximations, by use of
the word "about," it will be understood that the particular value
forms another embodiment. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
and independently of the other endpoint. As used herein the term
"about" refers to .+-.20%, preferably .+-.10%, and even more
preferable .+-.5%.
[0032] The present invention discloses isolated and propagated
human NSCs which are obtained from amniotic fluid that is taken
from a patient whose fetus has been diagnosed to have a neural tube
defect (NTD). These amniotic fluid-derived NSCs (AF-NSCs) form
neurospheres and undergo long-term expansion in vitro.
Additionally, they express NSCs-specific markers including Nestin,
Sox2, Musashi-1 and ATP-binding cassette G2 (ABCG2) and also
exhibit telomerase activity. After they are induced to
differentiate in vitro, AF-NSCs display typical morphological
patterns and express specific markers that are consistent with
neurons, astrocytes, oligodendrocytes and dopaminergic neurons.
Furthermore, AF-NSCs can be grafted into an animal to treat
neurological disorders.
[0033] Therefore, the present invention provides a method for
obtaining isolated human neural stem cells, which comprises:
[0034] (a) collecting the cells from amniotic fluid obtained from a
pregnant human subject whose fetus has been diagnosed to have a
neural tube defect (NTD);
[0035] (b) incubating the cells with a culture medium; and
[0036] (c) isolating the human neural stem cells from the culture
medium,
[0037] wherein the isolated human neural stem cells express Nestin,
Sox2, Musashi-1 and ATP-binding cassette G2 (ABCG2) markers and
exhibit telomerase activity but do not express SSEA-3, SSEA-4,
TRA-1-60 and TRA-1-81.
[0038] A neural stem cell is an undifferentiated neural cell that
can be induced to proliferate. The neural stem cell is capable of
self-maintenance, meaning that with each cell division, one
daughter cell also be a stem cell. Therefore, the phrase "neural
stem cell" as used herein shall be understood to include, whenever
appropriate, true stem cells as well as neural progenitor
cells.
[0039] According to the invention, the neural tube defects (NTDs)
include, but are not limited to, anencephaly and myelomeningocele.
More specifically, the NTD is anencephaly.
[0040] In step (a) of the invention, the cells may be collected by
any method known in the art. For example, centrifugation and/or
filtration of the amniotic fluid can be used.
[0041] According to the invention, the culture medium represents
any medium that allows the neural stem cells to proliferate. The
culture medium may be, but is not limited to, NeuroCult NS-A
Proliferation medium (StemCell Technologies, Vancouver, BC,
Canada), Stemline.TM. Neural Stem Cell Expansion Medium (Sigma) (20
ng/ml epidermal growth factor (Sigma) and 10 ng/ml leukaemia
inhibitory factor (Chemicon) are additionally added), NS-A medium
(Euroclone) (1.times.N2 and 10 ng/ml each of EGF (PeproTech) and
bFGF are additionally added), NS-A basal serum-free medium
(Euroclone) (20 human recombinant EGF ng/ml, 10 ng/ml human
recombinant FGF2, 2 mM L-glutamine, 0.6% glucose, 9.6 .mu.g/ml
putrescine, 6.3 ng/ml progesterone, 5.2 ng/mlsodium selenite, 0.025
mg/ml insulin, and 0.1 mg/ml trans-ferrin (sodium salt, grade II;
Sigma; control medium) are additionally added), Dulbecco's minimal
essential medium (DMEM)/F12 (1:1) (B27 supplementation (1:50), 2 mM
glutamine, 50 units/ml penicillin 50 .mu.g/ml streptomycin (Gibco),
20 ng/ml human recombinant EGF, and 20 ng/ml bFGF (R&D Systems)
are additionally added), and Dulbecco's modified Eagle's medium
(DMEM)/HAMS-F12 (3:1, Gibco) (penicillin
G/streptomycin/amphotericin B (1% v/v; Gibco), B27 supplement (2%
v/v, Gibco), epidermal growth factor (EGF, 20 ng/ml, Sigma), and
fibroblast growth factor 2 (FGF-2, 20 ng/ml, R&D Systems) with
heparin (5 mg/ml, Sigma) are additionally added).
[0042] The present invention also provides isolated human neural
stem cells which have all of the same characteristics as those of
the neural stem cells obtained from the method of the present
invention. The cells of the invention are of use as a source of
cells for cell therapy. For example, the cells can be transplanted
to restore damaged neural circuitry and/or restore brain
function.
[0043] In certain embodiments, the isolated cells are present
within a pharmaceutical composition. Accordingly, the
pharmaceutical composition comprising the neural stem cells further
comprises a pharmaceutically acceptable carrier, such as one or
more buffers (neural buffered saline or phosphate buffered saline),
solutes that render the formulation isotonic, hypotonic or weakly
hypertonic with the blood of a recipient, suspending agents,
thickening agents and/or preservatives.
[0044] In some embodiments, the pharmaceutical composition is
formulated in a unit dosage injectable form, such as a solution,
suspension, or emulsion. Pharmaceutical compositions suitable for
injection of cells typically are sterile aqueous solutions and
dispersions. Carriers of injectable formulations can be a solvent
or dispersing medium containing, for example, water, saline,
phosphate buffered saline, polyol (e.g., glycerol, propylene
glycol, liquid polyethylene glycol, and the like), and suitable
mixtures thereof.
[0045] The skilled artisan can readily determine the amount of
cells and optional additives, vehicles, and/or carrier in the
pharmaceutical composition of the invention. Typically, any
additives (in addition to the cells) are present in an amount of
about 0.001 to about 50 wt % in solution, such as in phosphate
buffered saline. The active ingredient is present in the order of
micrograms to milligrams, such as about 0.0001 to about 5 wt %,
preferably about 0.0001 to about 1 wt %, and most preferably about
0.0001 to about 0.05 wt %; or about 0.001 to about 20 wt %,
preferably about 0.01 to about 10 wt %, and most preferably about
0.05 to about 5 wt %.
[0046] Neural stem cells can be used for transplantation into a
heterologous, autologous, or xenogeneic host. The neural stem cell
progeny can be administered to any animal with abnormal
neurological or neurodegenerative symptoms obtained in any manner,
including those obtained as a result of mechanical, chemical, or
electrolytic lesions, as a result of ischemia or hypoxia of neural
areas, or as a result of aging processes.
[0047] Therefore, the present invention also relates to a method
for treating a neurological condition in a mammal in need thereof,
which comprises administering the pharmaceutical composition of the
present invention to the mammal.
[0048] The terms "treating" and "treatment" mean the slowing,
interrupting, arresting or stopping of the progression of the
disease or condition and do not necessarily require the complete
elimination of all disease symptoms and signs. "Preventing" or
"prevention" is intended to include the prophylaxis of the
neurological disease, wherein "prophylaxis" is understood to be any
degree of inhibition of the time of onset or severity of signs or
symptoms of the disease or condition, including, but not limited
to, the complete prevention of the disease or condition.
[0049] Neurological conditions that can be treated according to the
present invention can be generally classified into three classes:
those diseases with ischemic or hypoxic mechanisms;
neurodegenerative diseases; and neurological and psychiatric
diseases associated with neural cell death. Other neurological
conditions that can be treated according to the present invention
also include enhancing cognitive ability and the treatment of brain
tumors, such as glioblastomas, astrocytomas, meningiomas, and
neurinomas.
[0050] Diseases with ischemic or hypoxic mechanisms can be further
subclassified into general diseases and cerebral ischemia. Examples
of such general diseases involving ischemic or hypoxic mechanisms
include myocardial infarction, cardiac insufficiency, cardiac
failure, congestive heart failure, myocarditis, pericarditis,
perimyocarditis, coronary heart disease (stenosis of coronary
arteries), angina pectoris, congenital heart disease, shock,
ischemia of extremities, stenosis of renal arteries, diabetic
retinopathy, thrombosis associated with malaria, artificial heart
valves, anemias, hypersplenic syndrome, emphysema, lung fibrosis,
and pulmonary edema. Examples of cerebral ischemia disease include
stroke (as well as hemorrhagic stroke), cerebral microangiopathy
(small vessel disease), intrapartal cerebral ischemia, cerebral
ischemia during/after cardiac arrest or resuscitation, cerebral
ischemia due to intraoperative problems, cerebral ischemia during
carotid surgery, chronic cerebral ischemia due to stenosis of
blood-supplying arteries to the brain, sinus thrombosis or
thrombosis of cerebral veins, cerebral vessel malformations, and
diabetic retinopathy.
[0051] Examples of neurodegenerative diseases include amyotrophic
lateral sclerosis (ALS), Parkinson's disease, Huntington's disease,
Wilson's disease, multi-system atrophy, Alzheimer's disease, Pick's
disease, Lewy-body disease, Hallervorden-Spatz disease, torsion
dystonia, hereditary sensorimotor neuropathies (HMSN),
Gerstmann-Straussler-Schanker disease, Creutzfeld-Jakob-disease,
Machado-Joseph disease, Friedreich ataxia, non-Friedreich ataxias,
Gilles de la Tourette syndrome, familial tremors,
olivopontocerebellar degenerations, paraneoplastic cerebral
syndromes, hereditary spastic paraplegias, hereditary optic
neuropathy (Leber), retinitis pigmentosa, Stargardt disease, and
Kearns-Sayre syndrome.
[0052] Examples of neurological and psychiatric diseases associated
with neural cell death include septic shock, intracerebral
bleeding, subarachnoidal hemorrhage, multiinfarct dementia,
inflammatory diseases (such as vasculitis, multiple sclerosis, and
Guillain-Barre-syndrome), neurotrauma (such as spinal cord trauma,
and brain trauma), peripheral neuropathies, polyneuropathies,
epilepsies, schizophrenia, depression, metabolic encephalopathies,
and infections of the central nervous system (viral, bacterial,
fungal).
[0053] Since the human neural stem cells isolated by the present
invention can form neurospheres, undergo long-term expansion and
differentiate into astrocytes, oligodendrocytes and dopamineric
neurons in vitro, the isolated human neural stem cells are suitable
for drug discovery or neurotoxicity test.
[0054] Therefore, the present invention further relates to an in
vitro method for screening drug candidates, which comprises
contacting the isolated human neural stem cells with a drug
candidate; and determining one or more cell conditions of the
cells; if the determined one or more cell conditions are better
than the same condition(s) of the cells without contacting the drug
candidate, it represents that the drug candidate has potential in
the treatment of neurological conditions.
[0055] In addition, a cytotoxic testing method may be included in
the present invention. The method is conducted in vitro and
comprises the steps of contacting the isolated human neural stem
cells with a drug candidate; and determining one or more cell
conditions of the cells; if the determined one or more cell
conditions are poorer than the same condition(s) of the cells
without contacting the drug candidate, it represents that the drug
candidate has cytotoxicity.
[0056] According to the invention, the isolated human neural stem
cells may be provided in the form of a neurosphere culture or a
monolayer culture; and the drug candidate to be tested may be an
organic or inorganic chemical, a peptide, a polypeptide or a
protein; and the cell condition determined may include, but is not
limited to, neurosphere formation, apoptosis, proliferation,
differentiation, migration, or any combination thereof.
[0057] The cell conditions may be determined by any method known in
the art for observing the changes of phenotype and/or genotype of
cells. For example, neurosphere formation, apoptosis,
proliferation, differentiation and migration may be observed by
using phase-contrast microscopy. Colorimetric and
immunofluorescent-based assay may also be used. Furthermore,
technologies such as gene expression, electrical activity
measurements and metabonomics can be used as tools. For example,
measurement of the level of mRNA of a cell marker which represents
a specific stage of cell development and maturation can be
used.
[0058] According to the invention, the term "better" means that the
level of cell condition (e.g., cell count, neurosphere diameter or
metabolic activity measured over time) of the drug
candidate-contacted human neural stem cells is "higher" than that
from the non-drug candidate-contacted human neural stem cell; and
the term "poorer" means that the level of cell condition (e.g.,
cell count, neurosphere diameter or metabolic activity measured
over time) of the drug candidate-contacted human neural stem cells
is "lower" than that from the non-drug candidate-contacted human
neural stem cell. In one embodiment of the present invention, the
term "better" represents at least 0.5-fold higher, and preferably
at least 1-fold higher; and the term "poorer" represents 0.5-fold
lower, and preferably at least 1-fold lower.
[0059] The specific examples below are to be construed as merely
illustrative, and not limitative of the remainder of the disclosure
in any way whatsoever. Without further elaboration, it is believed
that one skilled in the art can, based on the description herein,
utilize the present invention to its fullest extent. All
publications cited herein are hereby incorporated by reference in
their entirety.
Examples
Materials and Methods
Sample Collection
[0060] The amniotic fluid samples used in this study were obtained
from Cathay General Hospital in Taipei and Chang Gung Memorial
Hospital in Taoyuan, Taiwan. Pregnant women aged 25 to 35 years
underwent amniotic fluid sampling performed for diagnostic purposes
between 16 and 20 weeks gestation. Fetuses were diagnosed with an
NTD of anencephaly or without an NTD by ultrasound and screening of
maternal serum. All procedures were approved by the Institutional
Review Boards of Cathay General Hospital and Chang Gung Memorial
Hospital, and all participants provided written informed consent to
participate in this study.
Cultivation of AF-NSCs
[0061] Each amniotic fluid sample was centrifuged at 1,000 rpm for
5 min, and the cell pellet was resuspended in NeuroCult.TM. NS-A
Proliferation medium (StemCell Technologies, Vancouver, BC, Canada)
in a T25 flask at 37.degree. C. in a 5% CO.sub.2 humidified
atmosphere. After 3-5 days, some attached neural-like cells could
be observed from the NTD samples, and the suspended cells and
debris were removed by changing the media. After adding fresh
NeuroCult NS-A proliferation medium, the attached neural-like cells
replicated and rounded-up to form primary neuroshperes. To maintain
these cells, 1/5 volume of additional culture medium was added
every 2-3 days. The initial mature neurospheres could be observed
3-4 weeks after plating. These cells were designated as human
amniotic fluid derived neural stem cells (AF-NSCs).
[0062] When the neurospheres grew to 50-100 .mu.m in diameter,
these cells were passaged. First, the cells were centrifuged at 800
rpm for 5 min and treated with TrypLE (Life Technologies,
Gaithersburg, Md., USA) at 37.degree. C. for 3 min. After an
additional centrifugation step to remove the TrypLE solution, the
cells were resuspended in the NeuroCult NS-A proliferation medium,
and a single cell suspension was obtained by pipetting carefully to
avoid bubble formation. AF-NSCs were seeded at a density of
0.5-1.times.10.sup.4/cm.sup.2 and were maintained as described
above. Neurospheres were passaged every 10-14 days, and the AF-NSCs
could be expanded for more than 8 months in vitro. The neurospheres
could also be cryopreserved for subsequent experiments.
[0063] To determine the optimal seeding density, the AF-NSCs were
plated at densities from 1,000-20,000 cells/cm.sup.2 in a T25 flask
and cultured as previously described. After 10 days, the
neurospheres were collected and trypsinized. Cell counts were
performed with a hemocytometer, and the doubling time per passage
was calculated. Each of the described experiments was performed in
triplicate.
[0064] In cumulative cell number test, AF-NSCs were seeded at 5,000
cells/cm.sup.2 in a T25 flask and cultured as previously described.
The cells were passaged every 10-14 days, and cells counts were
performed at each passage to calculate the fold increase in cells
along with the total cell number.
Flow Cytometry
[0065] AF-NSCs were trypsinized and resuspended as single cells in
phosphate buffered saline (PBS). For direct analysis, the cells
were fixed with Cytofix.TM. (BD Biosciences, San Jose, Calif., USA)
with or without permeabilization, and immunolabeled with the
following anti-human antibodies: CD73-phycoerythrin (PE),
CD105-Fluorescein isothiocyanate (FITC), CD117-PE, HLA-I-PE,
HLA-DR-PE, Nanog-PE, Oct-4-FITC, Sox2-PE, ABCG2-PE (all from BD
Biosciences), SSEA-1-PE, SSEA-3-FITC, SSEA-4-PE, TRA-1-60-PE,
TRA-1-81-PE, Nestin-FITC (all from R&D Systems, Minneapolis,
Minn., USA) or CD133-PE (Merck Millipore, Billerica, Mass., USA).
For indirect analysis, the cells were fixed, permeabilized with
Perm Buffer II (BD Biosciences), blocked, immunolabeled with
Musashi-1 (R&D Systems) and stained with an Alexa Fluor 488 dye
(Life technologies). All samples were processed using a FACSCantoII
flow cytometer (BD Biosciences), and at least 30,000 events were
captured per sample. The data acquisition and analysis were
performed using FACSDiva 6.0 (BD Biosciences) and FCS Express V3.00
(De Novo Software, Thornhill, Canada).
Immunocytochemistry
[0066] The cells were fixed with 4% paraformaldehyde (Merck
Millipore) and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich,
St Louis, Mo.). After being blocked with 10% specific normal serum
in PBS for 30 min, the cells were incubated with the appropriate
primary antibodies: Tuj-1 (Sigma), Nestin, Sox2,
microtubule-associated protein 2 (MAP2), neural filament heavy
chain (NFH), glial fibrillary acidic protein (GFAP), human
neuron-specific nuclear protein (hNeuN), tyrosine hydroxylase (TH)
(all from Merck Millipore), Musashi-1, 04, or aromatic L-amino acid
decarboxylase (AADC) (all from R&D system) for 1 hr. After
being washed twice, the cells were then incubated with an Alexa
Fluor 488 or Alexa Fluor 546-conjugated secondary antibody (Life
Technologies) for 1 hr at room temperature. The resulting
immunoreactive cells were visualized under a confocal microscope
(TCS--SPS-X AOBS, Leica, Solms, Germany) or a fluorescent
microscope (Axio Observer.Z1, Carl Zeiss, Oberkochen, Germany).
Telomerase Activity Assay
[0067] Telomerase activity was measured by the telomeric repeat
amplification protocol (TRAP) using a commercially available
TRAPeze RT kit (Merck Millipore). The amplified TRAP reaction
products were separated on a 12.5% polyacrylamide gel and
visualized as TRAP ladder patterns.
Quantitative Polymerase Chain Reaction (qPCR)
[0068] Total RNA was extracted with the TRIzol Reagent (Invitrogen,
Carlsbad, Calif., USA), and first strand cDNA was synthesized
according to the manufacturer's protocol using M-MuLV Reverse
Transcriptase (Thermo Scientific, San Jose, Calif., USA) and an
oligo-dT primer. qPCR was performed with the SYBR Green PCR master
mix (Thermo Scientific) using the ABI Prism 7700 Sequence Detection
System (Applied Biosystems, Foster City, Calif.). The relative
expression level of .beta.-actin was used as an internal control to
normalize gene expression in each sample. Relative quantification
of marker genes was performed according to the .DELTA..DELTA.Ct
method. The primer pairs used in this study are listed in Table
2.
TABLE-US-00001 TABLE 2 Oligonucleotide primers used in this study.
SEQ ID Primer Sequence (5' to 3') No. Nestin Forward:
CCCTGACCACTCCAGTTTAG 1 Reverse: CCTCTATGGCTGTTTCTTTCTC 2 Sox-2
Forward: CCGGCACGGCCATTAAC 3 Reverse: CTCCCATTTCCCTCGTTTTTC 4 Oct-4
Forward: TGCAGGCCCGAAAGAGAAAG 5 Reverse: GATCTGCTGCAGTGTGGGTTT 6
Nanog Forward: TGCCTCACACGGAGACTGTCT 7 Reverse:
AGTGGGTTGTTTGCCTTTGG 8 hTERT Forward: AGCTATGCCCGGACCTCCAT 9
Reverse: GCCTGCAGCAGGAGGATCTT 10 Tuj-1 Forward:
AAGCCAGCAGTGTCTAAACCC 11 Reverse: GGGAGGACGAGGCCATAAATAC 12 MAP2
Forward: GTGACAAGGAGTTTCAAACAGGAA 13 Reverse:
CTGATGGATAACTCTGTGCGAGA 14 GFAP Forward: GCGAGGAGAACCGGATCAC 15
Reverse: TTCACCACGATGTTCCTCTTGA 16 CNP Forward:
CCCAGGGAGAAGATGGACTTG 17 Reverse: CTTTAACACATCTTGTTGAGCGTACTC 18
MBP Forward: AGGCAGAGCGTCCGACTATAAA 19 Reverse:
GACTATCTCTTCCTCCCAGCTTAA AA 20 O2 Forward: CGGCGTTCGGTATCAGA 21
Reverse: GAACGGCCACAGTTCTAAGAG 22 AADC Forward: GGACCACAACATGCTGCTC
23 Reverse: CACTCCATTCAGAAGGTGCC 24 Lmx-1b Forward:
CCGAAAGGTCCGAGAGACACT 25 Reverse: AGCTTCTTCATCTTTGCTCTTTGG 26 Pax2
Forward: CCTGACCCCTGGGCTTGAT 27 Reverse: GTATGTCTGTGTGCCTGACACGTT
28 Nurr-1 Forward: GGCGAACCCTGACTATCAAATG 29 Reverse:
GCCCCGGATGATCTCCAT 30 .beta.-actin Forward: TGTGGATCAGCAAGCAGGAGTA
31 Reverse: CAAGAAAGGGTGTAACGCAACTAAG 32
AF-NSC Differentiation
[0069] The AF-NSCs-derived neurospheres (passage #10-12) were
trypsinized and seeded in NeuroCult.TM. NS-A proliferation medium
at a density of 5.times.10.sup.4 cells/cm.sup.2 on 100 .mu.g/ml
poly-L-lysine- (Sigma-Aldrich) and 10 .mu.g/ml laminin-
(Sigma-Aldrich) coated culture dishes. Upon cell attachment to the
bottom of the dishes, neural differentiation was induced by the
addition of NeuroCult.TM. NS-A differentiation medium (StemCell
Technologies) according to the manufacture's protocol. After
induction, the differentiated cells were fixed with 4%
paraformaldehyde for immunocytochemistry or collected for mRNA
extraction and subsequent qPCR. To direct the differentiation of
specific cell types, AF-NSCs were seeded on plates coated with 100
.mu.g/ml poly-L-lysine at a density of 5.times.10.sup.4
cells/cm.sup.2 in the presence of NS-A proliferation medium. After
attachment, the medium could be changed to a specific induction
medium that would produce astrocytes, oligodendrocytes and
dopamineric neurons were according to the previously published
protocols [27].
Focal Ischemia and AF-NSCs Transplantation
[0070] All the Sprague-Dawley rats (8 wks old, 250-300 g) were
obtained from Lasco (Ilan, Taiwan) and housed in an animal facility
at National Chung Hsing University. All experimental procedures
were devised with the welfare of the animal in mind and were
approved by the Institutional Animal Care and Use Committee of
National Chung Hsing University. The rats (n=12) were subjected to
a 1.5 hr long suture occlusion of the middle cerebral artery (MCAO)
in the right hemisphere [28]. On the first post-operative day,
1.times.10.sup.6 AF-NSCs (passage #10-12, 10 .mu.L, 1 .mu.L/min)
were transplanted into the damaged striatum at the location of AP:
-0.4 R: 3.4 DV:5 (n=6). The animals were sacrificed 4 weeks after
the operation, and their brains were fixed with 4% paraformaldehyde
by transcardial perfusion.
Behavioral Assays
[0071] MCAO rats underwent both rotarod and grip strength assays.
All rats performed similarly on both pieces of equipment after one
week of training. For the rotarod test, sham-operated rats could
remain on the cylinder when it was accelerated from 4 to 40 rpm
within 300 s. To test their grip strength, the rats grasped the
pull bar with their forepaws, and their grip strength was
quantified by an electronic sensor that was connected to the bar.
The results of three trials for each rat were recorded.
TTC Staining and Immunohistochemistry
[0072] MCAO rats brains were sectioned coronally into 6 slices,
which were then immersed with 2% 2,3,5-triphenyltetrazolium
chloride (TTC) for 30 minutes at 37.degree. C. followed by formalin
fixation. Infarcted areas were pale, while normal brain tissue was
stained red. For comparison with the area of healthy hemisphere,
the residual portion of stroke hemisphere in which the pale
infracted, the hollow liquidation and the normal tissue region had
diminished was assigned as the atrophy area. The infarcted and
atrophied areas were estimated with a computer image analysis
system (Image-Pro Plus, Media Cybernetics, Carlsbad, Calif., USA),
and the extent of tissue damage was calculated as a percentage of
the total area of the contralateral healthy hemisphere.
[0073] For the immunohistochemical analysis, the brains were
dehydrated with a sucrose gradient, embedded with OCT (Sakura Fine
Technical, Tokyo, Japan), frozen at -70.degree. C., and
cryosectioned at a thickness of 40 .mu.m. For immunostaining, the
sections were rinsed in PBS containing 0.1% Tween-20 (PBST),
permeabilized with 0.1% Triton X-100, and blocked with 10% specific
normal serum in PBS for 30 min prior to overnight incubation with
the primary antibodies, including the human Nuclei (Merck
Millipore) and Nestin (Merck Millipore). Next, the sections were
washed twice with PBST and incubated with the appropriate
rhodamine- or FITC-conjugated secondary antibody (Thermo
Scientific) for 1 hr at room temperature. Finally, a fluorescent
microscope was used to visualize the distribution of labeled
cells.
Statistical Analysis
[0074] All results in the examples are presented as
mean.+-.standard deviation (SD). Significant differences between
the two mean values were compared using the Student's t-test.
One-way ANOVA with Scheffe's post hoc test was used to assess
significant differences if more than two groups were compared. The
results were considered statistically significant when
p<0.05.
Example 1. Isolation of NSCs from Amniotic Fluid
[0075] To isolate NSCs from amniotic fluid, normal (n=7) and NTD
(anencephaly n=6, non-anencephaly n=6)-derived amniotic fluid
specimens were collected by amniocentesis. When the cells isolated
from these samples were cultured in NeuroCult NS-A proliferation
medium, only a subset of the anencephaly samples produced some
slightly attached neural-like cells and colonies during the first
3-5 days (FIG. 1A). After careful removal of the unattached cells
and cellular debris, these neural-like cells proliferated and
rounded-up to form primary neuroshperes that grew in suspension
after 3 weeks (FIG. 1A). Upon passaging, the neurospheres were
trypsinized into single cells, and cells proliferated and reformed
neurospheres in suspension with each passage (FIG. 1B). The
diameter of the neurospheres ranged from approximately 50 to 100
.mu.m, and they possessed the classic microspikes on their outer
surface (FIG. 1B). These AF-NSCs could be expanded by continual
passaging. It is important to note that AF-NSCs could only be
established from amniotic fluid samples taken from NTD patients
diagnosed with anencephaly (Table 1). AF-NSC lines could be
established from 4 out of the 6 anencephaly samples (success rate
67%), and all 4 lines had a normal karyotype (FIG. 6).
TABLE-US-00002 TABLE 1 Sources and outcome of amniotic fluid
samples. Total # of AF-NSCs Success Patient Status samples obtained
rate (%) No defect 7 0 0 NTD anencephaly 6 4 67 Non-anencephaly 6 0
0
[0076] The rate of neurosphere growth was influenced by the density
at which the AF-NSCs were initially seeded. The optimal seeding
density was determined to be 5,000-10,000 cells/cm.sup.2 (FIG. 1C),
with almost zero growth occurring at a density below 2,500
cells/cm.sup.2. The AF-NSCs doubled at a rate of 109.4.+-.14.8 hrs
when they were plated at a density under 10,000 cells
cells/cm.sup.2.
[0077] To test the long-term expansion potential of the AF-NSCs, we
grew 2 different lines (starting at passage 5) in vitro over the
course of several months. Both lines maintained constant growth for
at least 5 months (FIG. 1D), and one line could be propagated for
over 8 months. These two cell lines could be expanded over
10.sup.5-10.sup.10-fold.
Example 2. Characterization of the AF-NSCs
[0078] To characterize the AF-NSCs, immunocytochemistry and flow
cytometry were used to detect the expression NSC-specific markers.
Confocal microscopy revealed that AF-NSC-derived neurospheres
strongly expressed both Nestin and Musashi-1 within cytoplasm.
Sox2, a nucleus protein, could also be observed in the neurospheres
(FIG. 2A).
[0079] Flow cytometry revealed that AF-NSCs express NSC-specific
markers Nestin, Sox2, Musashi-1 and ABCG2 (FIG. 2B). CD133 was
initially expressed at a low level, but the intensity of the signal
increased with subsequent passages. We also analyzed the expression
of embryonic stem (ES) cell-specific markers and determined that
our AF-NSCs expressed a similar set of the transcription factors to
ES cells, including Nanog, Oct-4, and Sox2, and low levels of
SSEA-1; however, they did not express SSEA-3, SSEA-4, TRA-1-60, and
TRA-81. Furthermore, the pattern of human leukocyte antigen (HLA)
expression in AF-NSCs was similar to most stromal cells, which
express HLA class I but not HLA class II. When compared to
mesehchymal stem cells from amniotic fluid, which express CD73,
CD105 and occasionally CD117, our AF-NSCs did not express either
CD105 or CD117 and only occasionally expressed CD73. Interestingly,
the expression pattern of all markers which we detected was
maintained in AF-NSCs throughout their time in vitro (through
passage #20-22).
[0080] We also employed qPCR to measure the expression levels of
these markers throughout multiple passages and found that Nestin,
Sox2, Oct-4, Nanog and hTERT were all consistently expressed over
the course of 20 passages (FIG. 2C), which confirmed our previous
observations with flow cytometry. Finally, we assessed the
telomerase activity of our AF-NSCs and determined that the activity
levels were maintained despite increasing passage numbers in
long-term cultures (FIG. 2D).
Example 3. In Vitro Neural Differentiation of AF-NSCs
[0081] To determine whether AF-NSCs could differentiate into
neurons, the cells were dissociated into a single cell suspension,
cultured in NeuroCult.TM. differentiation medium and subsequently
analyzed by immunocytochemistry. After 2 days of induction, the
AF-NSCs began to undergo morphological changes, and only Nestin and
Tuj-1 were expressed within cells at this stage (FIG. 3A).
Additional neuron-specific markers, including Nestin, Tuj-1, MAP2,
hNeuN and NFH, could be detected after 7 days of induction (FIG.
3B). We also used qPCR to determine the transcription levels of
these neuron-specific genes and found that Tuj-1 and MAP2 were
upregulated by 5.2- and 6.2-fold, respectively, after 7 days of
differentiation. In contrast, the expression of Nestin, a
NSC-specific gene, significantly decreased during this same time
period (FIG. 3C).
[0082] Next, we induced our AF-NSCs into astrocytes,
oligodendrocytes and dopaminergic neurons by exposing them to
defined differentiation media. Most (>80%) AF-NSCs could be
induced to become GFAP positive astrocytes (FIG. 4A) as determined
by the dramatic 4,700-fold increase in GFAP expression after 2
weeks of induction (FIG. 4B). Oligodendrocytes were detected by
immunostaining for O4 antigen (FIG. 4A). Compared with
undifferentiated AF-NSCs, induction in defined medium caused the
expression of the oligodendrocyte-specific genes CNP, MBP and O2 to
increase to 2.15-, 4.97- and 1.9-fold, respectively. Moreover, the
presence of TH- and AADC-positive cells after 1 month suggested
that AF-NSCs could give rise to dopaminergic neurons (FIG. 4A).
This observation was verified by qPCR analysis, which determined
that markers specific for dopaminergic neuron, including AADC,
Pax2, Lmx-1b and Nurr-1, were significantly upregulated after
differentiation (FIG. 4B).
Example 4. AF-NSCs were Transplanted into Ischemia Rats and Induced
Functional Recovery
[0083] To determine whether AF-NSCs could induce functional
recovery from stroke in vivo, 1.times.10.sup.6 cells were
transplanted into the ischemic boundary zone of a rat brain that
had undergone MCAO. Non-treated rats with an MCAO consistently
showed impaired motor performance when compared to healthy control
as assessed by the rotarod and grip strength tests.
[0084] In the rotarod test, the MCAO group that was treated with
AF-NSCs experienced a 43.+-.18% reduction in time when compared to
sham-injected MCAO control rats, which suggests that the presence
of AF-NSCs improved the motor deficits observed in ischemic rats.
Notably, the AF-NSCs promoted rapid recovery of motor function
during the first week post-injection (85.+-.26%), and this
improvement was maintained for 4 weeks post-engraftment (98.+-.18%;
FIG. 5A).
[0085] A similar improvement in function was observed with the grip
strength test (FIG. 5B). Experiencing an MCAO significantly
decreased the rat's maximum grip strength. One week after they
received a transplant of AF-NSCs, the maximum grip strength of
these ischemic rats was similar to that of the sham-operated
control group. However, the rats that received AF-NSCs could
recover their normal grip strength 3 to 4 weeks
post-transplantation, whereas the sham-operated controls remained
weak.
[0086] To determine the extent of damage after MCAO, the engrafted
brains were stained with TTC 4 weeks after the AF-NSCs transplant.
The size of the infarcted area was significantly reduced after
transplantation (sham: 22.17.+-.1.12%; AF-NSCs transplanted:
0.62.+-.0.15%), whereas the atrophied region remained the same
(sham: 7.21.+-.0.48%; AF-NSCs transplanted: 5.26.+-.0.57%; FIG.
5C). We then used immunohistochemistry to determine whether the
AF-NSCs survived in the rat's brain after being transplanted by
double labeling cryosections with antibodies against Nestin and
human Nuclei antibodies. Our results showed that the Nestin/human
nuclei double positive cells were located close the injection area
(FIG. 5D), which suggests that the engrafted AF-NSCs could survive
and integrated into the host brain.
DISCUSSION
[0087] Over the past two decades, primary neural stem cells have
been isolated from both the fetal and adult CNS. Recent studies
have suggested that cells with the potential to become neurons can
also be found in amniotic fluid [23, 25, 29]; however, human neural
stem cells have not yet been isolated and cultured from this
location. Turner et al. reported that NSCs could be isolated and
cultured from amniotic fluid of Sprague-Dawley rats that had
undergone prenatal exposure to retinoic acid that resulted in NTDs;
however, these cells were absent in healthy control rats [26].
These AF-derived cells exhibited typical neural progenitor
morphology and robustly expressed the NSC-specific markers Nestin
and Sox-2. Previous studies have also reported that there is a
statistically significant increase in the number of neural stem
cells that can be derived from the amniotic fluid of rats with
spina bifida when compared to those animals with exencephaly alone
or with both spina bifida and exencephaly [30]. In this study, we
were able to isolate AF-NSCs from human amniotic fluid collected
from fetuses that had been diagnosed with anencephaly (67%) but not
from normal patients or from those with a non-anencephaly NTD.
These results suggest that the proportion of neural stem cells in
amniotic fluid is higher in those fetuses with an NTD, specifically
anencephaly. Anencephaly occurs when the anterior neural tube fails
to close and results in a total or partial absence of the cranial
vault and cerebral hemispheres. The outward flow of cerebrospinal
fluid into the amniotic fluid was also used in diagnosing an NTD
[30-32]. We argue that the presence of neural stem cells in the
amniotic fluid could be caused by leakage from the exposed neural
tissues and/or the cerebrospinal fluid into the amniotic
cavity.
[0088] Neural stem cells can self-renew and differentiate into all
CNS cell types and are typically grown as neurospheres under
serum-free conditions in vitro [33]. In our study, all 4 AF-NSC
lines that we established could be maintained as neurospheres for
more than 5 months and developed typical microspikes on their
surface. Previous studies have suggested that neural precursor
cells isolated from human embryo have decreased telomerase
activity, which declines to an undetectable level after 20
population doublings [34]. Conversely, our AF-NSCs could be
expanded over several months and maintained their telomerase
activity even at later passage numbers. However, although they
maintained their telomerase activity, AF-NSCs did not form
teratomas in vivo. Typical human NSCs cells isolated from various
CNS structures in 6-14.5 week-old human fetuses experienced a long
doubling time (8-10 days) in vitro [35]. Comparatively, our AF-NSCs
divided every 4-5 days. Because the growth of NSCs is affected by
cell density due to the additional cell-cell interactions, the
number of neurospheres is greater at higher cell density than at
low cell density [36, 37]. We found that AF-NSCs remained in a
quiescent state (<1 doubling/passage) when seeded at low
densities (<2,500 cells/cm.sup.2), whereas they would continue
to proliferate under conditions of higher cell density cultures
(5,000-10,000 cells/cm.sup.2). However, the cells could not grow
when the density was too high (>20,000 cells/cm.sup.2).
[0089] Nestin, Sox2, ABCG2, SSEA-1 and Musashi-1 have all been
previously established as NSC-specific markers [38, 39]. Our
AF-NSCs maintained their expression of these markers during
long-term cultivation in vitro. CD133 is a cell surface marker used
to isolated hematopoietic stem cells and neural stem cells [40]. In
this study, the expression of CD133 increased in the AF-NSCs
throughout their time in culture. Amniotic fluid has been
identified as an ideal source to isolate fetal multipotent stem
cells (AFSCs) [24, 41]. AFSCs share some markers with mesenchymal
stem cells, such as CD73, CD105 and CD117, and with pluripotent
stem cells, such as Oct-4, Nanog, and Sox2 etc. However, AF-NSCs
did not express either CD105 or CD117, and only expressed CD73 on
their surface. While AF-NSCs did express Nanog and Oct-4, they did
not stain for any embryonic stem cell markers, including SSEA-3,
SSEA-4, TRA-1-60 and TRA-1-81. Taken together, these results
suggest that AF-NSCs are distinct from previously identified
AFSCs.
[0090] Another hallmark of NSCs is their ability to differentiate
into multiple CNS cell types. We have demonstrated that AF-NSCs
have the potential to become neurons, astrocytes, oligodendrocytes,
and dopaminergic neurons in vitro. During oligodendrocyte
development, O4 is expressed in both oligodendrocyte precursors and
mature oligodendrocytes, whereas CNP and MBP are only expressed in
mature myelinating oligodendrocytes [42]. AF-NSCs could be induced
into O4 immunoreactive cells successfully in vitro; however, these
cells did not express either CNP or MBP at the protein level (data
not shown), although a significant increase in mRNA could be
detected by qPCR in multiple AF-NSC lines. This discrepancy may
reflect a need for further stimulation in vitro to produce mature
oligodendrocytes. Previous studies have demonstrated that
brain-derived NSCs can differentiate into neurons and provide
functional improvement in the behavior of ischemic rats [43-44].
Like fetal NSCs, undifferentiated AF-NSCs can be grafted
efficiently near the lesioned region in a rat stroke model.
Furthermore, AF-NSCs can induce a recovery of the ischemia-induced
reduction in grip-strength and rotarod performance. One hypothesis
is that NSC transplantation might induce tissue repair because
these cells attenuate inflammation, increase anti-apoptotic
activity or reduce infarct size [43, 45]. Here, we observed a
marked reduction of infarct size in ischemic rats after the
injection of AF-NSCs. Taken together, these results suggest that
the AF-NSCs not only have the potential to differentiate in vitro
but also have a neuroprotective effect on ischemic rats.
[0091] The main obstacles in using human NSCs for cellular therapy
have typically been the limitation of available sources,
complicated isolation protocols and time-consuming expansion. In
this study, we have demonstrated AF-NSCs derived from patients with
an NTD can be isolated and expanded ex vivo and exhibit similar
physiological characters to other sources of NSCs. Using high
resolution ultrasonography and amniocentesis to detect an NTD
during pregnancy would allow human AF-NSCs to be efficiently
collected. Therefore, human AF-NSC banks could be established for
clinical and preclinical testing purposes.
CONCLUSION
[0092] In conclusion, AF-NSCs derived from fetuses with NTDs share
common characteristics with other sources of human NSCs, including
the ability to from neurospheres, expression of stem cell-specific
makers, the ability to undergo long-term proliferation, the
potential to differentiate in vitro and the potential to have
therapeutic effects in a rat stroke model. Therefore, this novel
source of human NSCs could have a significant impact on future
translational and therapeutic studies.
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Sequence CWU 1
1
32120DNAArtificial SequencePrimer 1ccctgaccac tccagtttag
20222DNAArtificial SequencePrimer 2cctctatggc tgtttctttc tc
22317DNAArtificial SequencePrimer 3ccggcacggc cattaac
17421DNAArtificial SequencePrimer 4ctcccatttc cctcgttttt c
21520DNAArtificial SequencePrimer 5tgcaggcccg aaagagaaag
20621DNAArtificial SequencePrimer 6gatctgctgc agtgtgggtt t
21721DNAArtificial SequencePrimer 7tgcctcacac ggagactgtc t
21820DNAArtificial SequencePrimer 8agtgggttgt ttgcctttgg
20920DNAArtificial SequencePrimer 9agctatgccc ggacctccat
201020DNAArtificial SequencePrimer 10gcctgcagca ggaggatctt
201121DNAArtificial SequencePrimer 11aagccagcag tgtctaaacc c
211222DNAArtificial SequencePrimer 12gggaggacga ggccataaat ac
221324DNAArtificial SequencePrimer 13gtgacaagga gtttcaaaca ggaa
241423DNAArtificial SequencePrimer 14ctgatggata actctgtgcg aga
231519DNAArtificial SequencePrimer 15gcgaggagaa ccggatcac
191622DNAArtificial SequencePrimer 16ttcaccacga tgttcctctt ga
221721DNAArtificial SequencePrimer 17cccagggaga agatggactt g
211827DNAArtificial SequencePrimer 18ctttaacaca tcttgttgag cgtactc
271922DNAArtificial SequencePrimer 19aggcagagcg tccgactata aa
222026DNAArtificial SequencePrimer 20gactatctct tcctcccagc ttaaaa
262117DNAArtificial SequencePrimer 21cggcgttcgg tatcaga
172221DNAArtificial SequencePrimer 22gaacggccac agttctaaga g
212319DNAArtificial SequencePrimer 23ggaccacaac atgctgctc
192420DNAArtificial SequencePrimer 24cactccattc agaaggtgcc
202521DNAArtificial SequencePrimer 25ccgaaaggtc cgagagacac t
212624DNAArtificial SequencePrimer 26agcttcttca tctttgctct ttgg
242719DNAArtificial SequencePrimer 27cctgacccct gggcttgat
192824DNAArtificial SequencePrimer 28gtatgtctgt gtgcctgaca cgtt
242922DNAArtificial SequencePrimer 29ggcgaaccct gactatcaaa tg
223018DNAArtificial SequencePrimer 30gccccggatg atctccat
183122DNAArtificial SequencePrimer 31tgtggatcag caagcaggag ta
223225DNAArtificial SequencePrimer 32caagaaaggg tgtaacgcaa ctaag
25
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