U.S. patent application number 11/364454 was filed with the patent office on 2006-07-06 for differentiation of brain tissue.
This patent application is currently assigned to Cedars-Sinai Medical Center. Invention is credited to Moneeb Ehtesham, Peter Kabos, John S. Yu.
Application Number | 20060148083 11/364454 |
Document ID | / |
Family ID | 26943536 |
Filed Date | 2006-07-06 |
United States Patent
Application |
20060148083 |
Kind Code |
A1 |
Yu; John S. ; et
al. |
July 6, 2006 |
Differentiation of brain tissue
Abstract
A method is described for generating a clinically significant
volume of neural progenitor cells from whole bone marrow. A mass of
bone marrow cells may be grown in a culture supplemented with
fibroblast growth factor-2 (FGF-2) and epidermal growth factor
(EGF). Further methods of the present invention are directed to
utilizing the neural progenitor cells cultured in this fashion in
the treatment of various neuropathological conditions, and in
targeting delivery of cells transfected with a particular gene to
diseased or damaged tissue.
Inventors: |
Yu; John S.; (Los Angeles,
CA) ; Kabos; Peter; (Los Angeles, CA) ;
Ehtesham; Moneeb; (Nashville, TN) |
Correspondence
Address: |
DAVIS WRIGHT TREMAINE LLP
865 FIGUEROA STREET
SUITE 2400
LOS ANGELES
CA
90017-2566
US
|
Assignee: |
Cedars-Sinai Medical Center
Los Angeles
CA
|
Family ID: |
26943536 |
Appl. No.: |
11/364454 |
Filed: |
February 28, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10253759 |
Sep 24, 2002 |
|
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11364454 |
Feb 28, 2006 |
|
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60334957 |
Oct 25, 2001 |
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Current U.S.
Class: |
435/368 |
Current CPC
Class: |
A61P 35/00 20180101;
C12N 2501/115 20130101; A61P 25/00 20180101; C12N 2506/1353
20130101; A61P 43/00 20180101; A61K 48/00 20130101; C12N 2506/11
20130101; A61P 25/28 20180101; A61P 9/00 20180101; C12N 5/0623
20130101; A61P 9/10 20180101; C12N 2510/00 20130101; A61K 38/00
20130101; C12N 2501/11 20130101; A61K 35/12 20130101 |
Class at
Publication: |
435/368 |
International
Class: |
C12N 5/08 20060101
C12N005/08 |
Claims
1. A method to generate neural progenitor cells, the method
comprising: culturing brain tissue in a medium comprising
fibroblast growth factor-2 (FGF-2) and epidermal growth factor
(EGF) to produce neural progenitor cells.
2. (canceled)
3. The method of claim 1, wherein the brain tissue is obtained from
an adult mammal.
4. The method of claim 1, wherein the brain tissue is obtained from
a fetus.
5. (canceled)
6. The method of claim 1, wherein the medium is Dulbecco's modified
Eagle medium (DMEM).
7. The method of claim 1, wherein the medium further comprises a
supplement.
8. The method of claim 7, wherein the supplement is selected from
the group consisting of B27, N2, and combinations thereof.
9. (canceled)
10. The method of claim 1, wherein the medium further comprises an
additional compound is selected from the group consisting of
interleukin-3 (IL-3), stem cell factor-1 (SCF-1), sonic hedgehog
(Shh), fms-like tyrosine kinase-3 (Flt3) ligand, leukemia
inhibitory factor (LIF), and combinations thereof.
11. The method of claim 1, wherein the medium further comprises an
antibiotic.
12. The method of claim 11, wherein the antibiotic is selected from
the group consisting of penicillin, streptomycin, and combinations
thereof.
13. The method of claim 1, wherein clusters of the neural
progenitor cells develop in the medium, and the method further
comprises separating at least one cluster from the medium.
14. The method of claim 13, further including subculturing the at
least one cluster.
15. The method of claim 14, wherein subculturing the at least one
cluster further comprises culturing the at least one cluster in a
medium comprising FGF-2 and EGF.
16-52. (canceled)
53. Neural progenitor cells, produced by the process comprising:
providing brain tissue from a mammal; and culturing said brain
tissue in a medium comprising fibroblast growth factor-2 (FGF-2)
and epidermal growth factor (EGF) to produce the neural progenitor
cells.
54. The neural progenitor cells of claim 53, wherein the medium
further comprises a supplement selected from the group consisting
of B27, N2, and combinations thereof.
55. The neural progenitor cells of claim 53, wherein the medium
further comprises an additional compound selected from the group
consisting of interleukin-3 (IL-3), stem cell factor-1 (SCF-1),
sonic hedgehog (Shh), fms-like tyrosine kinase-3 (Flt3) ligand,
leukemia inhibitory factor (LIF), and combinations thereof.
56. The neural progenitor cells of claim 53, wherein clusters of
the neural progenitor cells develop in the medium, and the process
further comprises: separating at least one cluster from the medium;
and subculturing the at least one cluster in a medium comprising
FGF-2 and EGF.
57. A method to generate neural progenitor cells, the method
comprising: providing brain tissue from a mammal; and culturing
said brain tissue in Dulbecco's modified Eagle Medium (DMEM)
comprising B27, penicillin, streptomycin, fibroblast growth
factor-2 (FGF-2) and epidermal growth factor (EGF), wherein sphere
clusters of the neural progenitor cells develop in the medium, and
the method further comprises separating at least one sphere cluster
from the medium and subculturing the at least one sphere cluster in
a medium comprising FGF-2 and EGF to produce neural progenitor
cells.
58. A neural progenitor cell, produced by the process comprising:
providing brain tissue from a mammal; and culturing said brain
tissue in Dulbecco's modified Eagle Medium (DMEM) comprising B27,
penicillin, streptomycin, fibroblast growth factor-2 (FGF-2) and
epidermal growth factor (EGF), wherein sphere clusters of neural
progenitor cells develop in the medium, and the process further
comprises separating at least one sphere cluster from the medium
and subculturing the at least one sphere cluster in a medium
comprising FGF-2 and EGF to produce a neural progenitor cell.
Description
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn. 119(e) of provisional application Ser. No.
60/334,957, filed Oct. 25, 2001, the contents of which are hereby
incorporated by reference.
FIELD OF THE INVENTION
[0002] Embodiments of the present invention are directed to a
method for generating a clinically substantial volume of neural
progenitor cells from mammalian whole bone marrow. Further
embodiments of the present invention are directed to the treatment
of neurological disorders using neural progenitor cells cultured in
this fashion.
BACKGROUND OF THE INVENTION
[0003] Nearly every cell in an animal's body, from neural to blood
to bone, owes its existence to a stem cell. A stem cell is commonly
defined as a cell that (i) is capable of renewing itself; and (ii)
can give rise to more than one type of cell (that is, a
differentiated cell) through asymmetric cell division. F. M. Watt
and B. L. M. Hogan, "Out of Eden: Stem Cells and Their Niches,"
Science, 284, 1427-1430 (2000). Stem cells give rise to a type of
stem cell called progenitor cells; progenitor cells, in turn,
proliferate into the differentiated cells that populate the
body.
[0004] The prior art describes the development, from stem cell to
differentiated cells, of various tissues throughout the body. U.S.
Pat. No. 5,811,301, for example, the disclosure of which is hereby
incorporated by reference, describes the process of hematopoiesis,
the development of the various cells that comprise blood. The
process begins with what may be a pluripotent stem cell, a cell
that can give rise to every cell of an organism (there is only one
cell that exhibits greater developmental plasticity than a
pluripotent stem cell; this is a fertilized ovum, a single,
totipotent stem cell that can give rise to an entire organism when
implanted into the uterus). The pluripotent stem cell gives rise to
a myeloid stem cell. Certain maturation-promoting polypeptides
cause the myeloid stem cell to differentiate into precursor cells,
which in turn differentiate into various progenitor cells. It is
the progenitor cells that proliferate into the various lymphocytes,
neutrophils, macrophages, and other cells that comprise blood
tissue of the body.
[0005] This description of hematopoiesis is vastly incomplete, of
course: biology has yet to determine a complete lineage for all the
cells of the blood (e.g., it is has yet to identify all the
precursor cells between the myeloid stem cell and the progenitor
cells to which it gives rise), and it has yet to determine
precisely how or why the myeloid cell differentiates into
progenitor cells. Even so, hematopoiesis is particularly well
studied; even less is known of the development of other organ
systems. With respect to the brain and its development, for
example, U.S. Pat. No. 6.040,180, the disclosure of which is hereby
incorporated by reference, describes the "current lack of
understanding of histogenesis during brain development." U.S. Pat.
No. 5,849,553, the disclosure of which is hereby also incorporated
by reference, describes the "uncertainty in the art concerning the
development potential of neural crest cells."
[0006] The identification and isolation of stem cells has daunted
researchers for decades. To date, no one has identified an
individual neural stem cell or hematopoietic stem cell. F. H. Gage,
"Mammalian Neural Stem Cells," Science, 287, 1433-1488 (2000).
There are two principal difficulties. First, stem cells are rare.
In bone marrow, for example, where hematopoiesis occurs, there is
only one stem cell for every several billion bone marrow cells. G.
Vogel, "Can Old Cells Learn New Tricks?" Science, 287, 1418-1419
(2000). Second, and more importantly, researchers have been unable
to identify molecular markers which are unique to stem cells; to
the typical immunoassay, most stem cells look like any other cell.
Id. Compounding this problem is that primitive stem cells may be in
a quiescent state. As a result, they may express few molecular
markers. F. H. Gage, supra.
[0007] A method to effectively isolate stem cells and culture them
in clinically significant quantities would be of immense
importance. Researchers are already transplanting immature neurons,
presumed to contain neural stem cells, from human fetuses to adult
patients with neurodegenerative disease. The procedure has reduced
symptoms by up to 50% in patients with Parkinson's disease in one
study. M. Barinaga, "Fetal Neuron Grafts Pave the Way for Stem Cell
Therapies," Science, 287, 1421-1422 (2000). Many of the
shortcomings of this procedure, including the ethical and practical
difficulties of using material derived from fetuses and the
inherent complications of harvesting material from adult brain
tissue, could be addressed by using cultures of isolated stem
cells, or stem cells obtained from adult individuals. D. W. Pincus
et al., Ann. Neurol. 43:576-585 (1998); C. B. Johansson et al.,
Exp. Cell. Res. 253:733-736 (1999); and S. F. Pagano et al., Stem
Cells 18:295-300 (2000). However, the efficient and large-scale
generation of neural progenitor cells for use in the treatment of
neurological disorders has been a challenge.
[0008] Recent evidence has suggested that progenitor cells outside
the central nervous system and bone marrow cells in particular may
have the ability to generate either neurons or glia in vivo. J. G.
Toma et al., Nat. Cell Biol. 3:778-783 (2001); E. Mezey et al.,
Science 290:1779-1782 (2000); T. R. Brazleton et al., Science
290:1775-1779 (2000); and M. A. Eglitis et al., Proc Natl. Acad.
Sci. 94:4080-4085 (1997). Bone marrow stromal cells have also been
shown to be capable of differentiating into neurons and glia in
vitro after a complicated and time-consuming culture process
spanning several weeks. The generation of neural progenitor cells
from whole bone marrow has, however, not been reported.
SUMMARY OF THE INVENTION
[0009] The invention described herein provides an efficient method
of generating a clinically significant quantity of neural
progenitor cells. These neural progenitor cells may be generated
from bone marrow or other appropriate sources, and may be used to
treat a variety of conditions, particularly neuropathological
conditions. Owing to the neural progenitor cells' ability to track
diseased or damaged neural tissue and to further replace the lost
function of such tissue, the cells of the present invention are
particularly useful in the treatment of conditions wherein neural
tissue itself is damaged.
[0010] Still further embodiments of the present invention describe
the use of the neural progenitor cells to target the delivery of
various compounds to damaged or diseased neural tissue. Neural
progenitor cells may be caused to carry a gene that induces the
cells themselves to secrete such compounds, or to otherwise effect
the local production of such compounds by, for example, initiating
or promoting a particular biochemical pathway. Since the neural
progenitor cells that carry these genes may track diseased or
damaged neural tissue, delivery of the particular compound may be
correspondingly targeted to such tissue. A dual treatment effect is
accomplished when the neural progenitor cells both replace lost or
damaged neural tissue function while simultaneously effecting the
targeted delivery of a therapeutic compound.
BRIEF DESCRIPTION OF THE FIGURES
[0011] The file of this patent contains at least one drawing
executed in color. Copies of this patent with color drawing(s) will
be provided by the Patent and Trademark Office upon request and
payment of the necessary fee.
[0012] FIG. 1 depicts neural progenitor cells obtained from human
bone marrow in accordance with an embodiment of the present
invention. FIG. 1A depicts cells from whole bone marrow that, when
plated on poly-D-lysine, form a monolayer that gives rise to
distinct cellular spheres after four days in culture. FIG. 1B
depicts the spheres of FIG. 1A at higher magnification; cells may
be easily collected, sub-cultured, and propagated separately in the
presence of growth factors. FIG. 1C depicts that the spheres, once
differentiated, attach and cells start migrating outward (arrows
indicate migrating cells). FIG. 1D depicts that the formed spheres
detach from the bottom and afterwards remain free-floating.
[0013] FIG. 2 is executed in color and depicts neural progenitor
cells obtained from human bone marrow in accordance with an
embodiment of the present invention. FIGS. 2A and 2B indicate that
neurospheres (i.e., spheres derived from neural cells) and bone
marrow-derived spheres, respectively, were morphologically
indistinguishable. FIGS. 2C and 2D indicate that the pattern of
nestin expression (red) was similar both in neurospheres and bone
marrow derived spheres, respectively. Nuclei of cells appear blue
owing to being counterstained with 4',6-diamidino-2-phenylindole
(DAPI).
[0014] FIG. 3 is executed in color and depicts neural progenitor
cells obtained from human bone marrow in accordance with an
embodiment of the present invention. FIG. 3A indicates that the
bone marrow-derived spheres expressed the ectodermal marker
vimentin. As depicted in FIG. 3B, a weak staining for fibronectin
was also observed in the neural progenitor cells. As depicted in
FIG. 3C, bone marrow-derived spheres exhibit strong expression of
CD90, and, as depicted in FIG. 3D, the majority of the cells in
spheres exhibit nuclear expression of Neurogenin 1.
[0015] FIG. 4 is executed in color and depicts a differentiation of
bone marrow derived cells into neurons and glia in accordance with
an embodiment of the present invention. After plating on a
substrate in media devoid of growth factors, the bone
marrow-derived spheres attached, migrated away from the primary
site of attachment, and displayed multiple morphologies, as
depicted in FIG. 4A. FIGS. 4B and 4C depict neural progenitor cells
of the present invention expressing the glial cell marker glial
fibrillary acidic protein (GFAP) after eight and nine days of
differentiation, respectively (cellular nuclei counterstained with
DAPI). FIGS. 4D and 4E depict neural progenitor cells of the
present invention expressing the neuronal marker Neuron Specific
Enolase (NSE) after eight days of differentiation (cellular nuclei
counterstained with DAPI). Scattered cells also expressed the later
neuronal marker MAP2, as depicted in FIG. 4F. After transplantation
of the bone marrow derived spheres into the hippocampus of a
syngeneic animal, cells expressing NeuN were found, as depicted in
FIG. 4G. Some of these cells appeared to integrate into the
hippocampal structure, as depicted in FIG. 4H. FIGS. 4I, 4J and 4K
depict a similar differentiation of bone marrow derived cells, with
alternate antibodies used for immunocytochemistry. FIG. 4I depicts
the use of the oligodendrocyte marker CNPase (1:400 Sigma) at
40.times. magnification, while FIGS. 4J and 4K depict the use of
the neuronal marker NF200 (1:100 Chemicon) at 20.times. and
40.times. magnification, respectively.
[0016] FIG. 5 is executed in color and depicts a gene transfer to
neural progenitor cells using a .beta.-galactosidase gene-bearing
replication-deficient adenoviral vector in accordance with an
embodiment of the present invention.
[0017] FIG. 6 is executed in color and depicts neural progenitor
cells infected with green fluorescent protein (GFP) bearing double
herpes simplex virus type I in accordance with an embodiment of the
present invention.
[0018] FIG. 7 is executed in color and depicts neurospheres
generated from primary fetal brain culture in accordance with an
embodiment of the present invention. FIG. 7A depicts neural
progenitor cells grown into spherical aggregates. FIG. 7B depicts
nestin expression by these neurospheres (nuclei counterstained with
DAPI). Neurons expressed .beta.-III tubulin, astrocytes expressed
GFAP, and oligodendrocytes expressed NPase (FIGS. 7C, 7D, and 7E,
respectively). FIG. 7F depicts expression of .beta.-galactosidase
by neural progenitor cells infected in vitro with AdLacZ.
Magnification 400.times. for FIGS. 7B, 7C, 7D, and 7E; 100.times.
for FIGS. 7A and 7F.
[0019] FIG. 8 is executed in color and depicts an intra-arterial
delivery of neural progenitor cells into an experimentally induced
ischemic lesion in accordance with an embodiment of the present
invention. Single cells are distributed widely throughout the brain
tissue (FIG. 8A). Transplanted cells exhibit tropism for injured
basal ganglia (FIG. 8B; at 400.times. magnification).
[0020] FIG. 9 is executed in color and depicts neural progenitor
cells tracking tumor cells in vivo in accordance with an embodiment
of the present invention. FIG. 9A depicts a thin outgrowth of tumor
cells deep into adjacent normal brain. FIG. 9B depicts a direct
extension of tumor mass into adjacent tissue. FIG. 9C depicts a
migration of glioma cells away from the primary tumor bed along a
white matter tract. FIG. 9D depicts a tumor microsatellite
independent of a main tumor mass. FIG. 9E depicts a high power
photomicrograph of the microsatellite depicted in FIG. 9D; further
depicting .beta.-galactosidase-positive neural progenitor cells
interspersed with tumor cells. FIG. 9F shows an inoculation of
neural progenitor cells (left panel) and a tumor mass (right panel)
into which neural progenitor cells migrated from the opposite
hemisphere (inset box). Neural progenitor cells appear blue
(expressing .beta.-galactosidase), whereas tumor cells appear red
(hypercellular areas stained intensively with neural red). "T"
represents tumor mass, outgrowths, and microsatellites. Arrows
indicate disseminating neural progenitor cells closely following
migrating pockets of tumor.
[0021] FIG. 10 is executed in color and depicts intratumoral CD4+
and CD8+T-cell infiltration in accordance with an embodiment of the
present invention. FIG. 10A depicts a flow cytometry analysis
demonstrating intratumoral T-cell infiltration in brain tissue
treated with neural progenitor cells secreting IL-12 (left panel)
and 3T3-IL-12 (center panel), and a comparative lack of
infiltration in tissue treated with neural progenitor cells
secreting LacZ (right panel). CD4+ (left panel) and CD8+ (right
panel) intratumoral infiltration is depicted in tissue treated with
neural progenitor cells secreting 3T3-IL-12, LacZ, and IL-12 (FIGS.
10B, 10C, and 10D, respectively). Aggregates appeared along the
tumor/normal tissue boundary in tissue treated with neural
progenitor cells secreting IL-12 (FIG. 10D, arrows indicate
aggregates). FIG. 10E depicts a comparison of T-cell infiltration
in comparable outgrowths from a primary tumor bed for tissue
treated with neural progenitor cells secreting IL-12 and 3Y3-IL-12
(FIG. 10E, left and right panels, respectively). "T" designates
tumor and "N" designates normal brain tissue. Magnification
100.times. for FIGS. 10B, 10C, and 10D, and 200.times. for FIG.
10E.
[0022] FIG. 11 is executed in color and depicts transplantation of
neural progenitor cells expressing GFP into rat hippocampus in
accordance with an embodiment of the present invention. FIG. 11A
depicts a migration of transplanted cells (green). FIG. 11B depicts
individual cells expressing NSE (red) and GFP together with NSE
(yellow). Transplanted cells were stained for NSE and exhibit GFP
(green), NSE (red), and the merged image of green fluorescent
protein (GFP) and NSE (green and red) (FIGS. 11C, 11D, and 11E,
respectively). Magnification 100.times. for FIG. 11A; 630.times.
for FIG. 11B; and 200.times. for FIGS. 11C, 11D, and 11E.
[0023] FIG. 12 is executed in color and depicts neural progenitor
cells, stained for LacZ, seen in the tumor outgrowth migrating out
from the main tumor mass at 10.times. (FIG. 12A) and 40.times.
(FIG. 12B) magnification. The sections were counterstained with
hematoxylin.
DETAILED DESCRIPTION OF THE INVENTION
[0024] Methods of the present invention are based on adult bone
marrow as a viable alternative source of neural progenitor cells
that may be used in therapeutic strategies for a variety of
neuropathological conditions.
[0025] Any population of cells where neural progenitor cells are
suspected of being found may be used in accordance with the method
of the present invention. Such populations of cells may include, by
way of example, mammalian bone marrow, brain tissue, or any
suitable fetal tissue. Preferably, cells are obtained from the bone
marrow of a non-fetal mammal, and most preferably from a human.
U.S. Pat. Nos. 6,204,053 B1 and 5,824,489, the disclosures of which
are hereby incorporated by reference, identify additional sources
of cells that contain or are thought to contain stem cells; any of
these cells may be used in accordance with the methods of the
present invention.
[0026] In one embodiment of the present invention, a mass of cells
may be harvested or otherwise obtained from an appropriate source,
such as, by way of example, adult human bone marrow. The mass of
cells may thereafter be grown in a culture, and may be further
subcultured where desirable, to generate further masses of cells.
Any appropriate culture medium may be used in accordance with the
methods of the present invention, such as, by way of example,
serum-free Dulbecco's modified Eagle medium (DMEM)/F-12 medium.
[0027] The medium of the present invention may include various
medium supplements, growth factors, antibiotics, and additional
compounds. Supplements may illustratively include B27 supplement
and/or N2 supplement (both available from Invitrogen Corporation);
growth factors may illustratively include fibroblast growth
factor-2 (FGF-2), epidermal growth factor (EGF), and/or leukemia
inhibitory factor (LIF); and antibiotics may illustratively include
penicillin and/or streptomycin. In preferred embodiments of the
present invention, growth factors are included in an amount of from
about 15 ng/ml to about 25 ng/ml. Additional compounds suitable for
use in the present invention may include, but are in no way limited
to, interleukin-3 (IL-3), stem cell factor-1 (SCF-1), sonic
hedgehog (Shh), and fms-like tyrosine kinase-3 (Flt3) ligand. While
not wishing to be bound by any theory, it is believed that these
particular compounds may enhance the production of spheres in
accordance with the methods of the present invention. Additional or
substituted supplements, growth factors, antibiotics, and
additional compounds suitable for use with the methods of the
present invention may be readily recognized by one of skill in the
art, and these are contemplated as being within the scope of the
present invention. In a most preferred embodiment of the present
invention, a culture medium is DMEM/F-12 medium supplemented with
B27, and additionally includes 10 ng/ml of both FGF-2 and EGF, as
well as penicillin and streptomycin.
[0028] After a sufficient time period (generally from about three
to about six days), clusters of neural progenitor cells (e.g.,
spheres) may form in a culture medium in which stem cells obtained
as described above are included. Individual clusters of neural
progenitor cells may be removed from the medium and sub-cultured
separate from one another. Such separation may be repeated any
desirable number of times to generate a clinically significant
volume of neural progenitor cells. These neural progenitor cells
may be capable of differentiating into a variety of neural cells,
such as, astrocytes, neurons, and oligodendroglia.
[0029] As used herein, a "clinically significant volume" is an
amount of cells sufficient to utilize in a therapeutic treatment of
a disease condition, including a neuropathological condition.
Furthermore, as used herein, "treatment" includes, but is not
limited to, ameliorating a disease, lessening the severity of its
complications, preventing it from manifesting, preventing it from
recurring, merely preventing it from worsening, mitigating an
undesirable biologic response (e.g., inflammation) included
therein, or a therapeutic effort to effect any of the
aforementioned, even if such therapeutic effort is ultimately
unsuccessful.
[0030] The neural progenitor cells of the present invention possess
a host of potential clinical and therapeutic applications, as well
as applications in medical research. Two possible therapeutic
mechanisms include: (1) using the cells as a delivery vehicle for
gene products by taking advantage of their ability to migrate after
transplantation, and (2) using the cells to replace damaged or
absent neural tissue, thereby restoring or enhancing tissue
function.
[0031] As discussed in the ensuing Examples, and with reference to
the first therapeutic mechanism indicated above, the neural
progenitor cells of the present invention are capable of "tracking"
diseased or damaged tissue in vivo. The cells may therefore be used
to aid in the targeted delivery of various compounds useful in the
treatment of diseased or damaged tissue. Delivery of such compounds
may be accomplished by transfecting the cells with a gene that
induces the cell to, for example, constitutively secrete that
compound itself, or promote a biochemical pathway that effects a
local production of that compound.
[0032] Thus, in one embodiment of the present invention, neural
progenitor cells may be transfected with or otherwise caused to
carry a particular gene, as per any conventional methodology. Such
methodologies may include introducing a particular gene into the
neural progenitor cells as a plasmid, or, more preferably, using
somatic cell gene transfer to transfect the cells utilizing viral
vectors containing appropriate gene sequences. Suitable viral
vectors may include, but are in no way limited to, expression
vectors based on recombinant adenoviruses, adeno-associated
viruses, retroviruses or lentiviruses, although non-viral vectors
may alternatively be used. In a preferred embodiment of the present
invention, one employs adenovirus serotype 5 ("Ad5")-based vectors
(available from Quantum Biotechnology, Inc., Montreal, Quebec,
Canada) to deliver and express desirable gene sequences in the
neural progenitor cells of the present invention. Once caused to
carry the desired gene, the neural progenitor cells may be
implanted in or otherwise administered to a mammal.
[0033] By employing this therapeutic mechanism, the neural
progenitor cells of the present invention may be used to treat a
variety of pathological conditions; potentially any condition where
mammalian neural tissue is diseased or damaged to the point that
neural progenitor cells will track the same. In the area of
neuropathological disorders, this therapeutic modality may be used
in the treatment of numerous conditions, some examples of which may
include: brain tumors (e.g., by targeting the delivery of cytokines
or other agents that enhance the immune response, or by targeting
the delivery of compounds that are otherwise toxic to tumor cells);
brain ischemia (e.g., by targeting the delivery of neuroprotective
substances such as brain-derived neurotrophic factor (BDNF), nerve
growth factor (NGF), and neurotrophin-3, -4, and -5 (NT-3, NT-4,
NT-5)); spinal cord injury (e.g., again, by targeting the delivery
of neuroprotective substances, or by targeting the delivery of
substances inducing neurite growth such as basic fibroblast growth
factor (bFGF), insulin-like growth factor-1 (IGF-1), and
glial-derived neurotrophic factor (GDNF)); and neurodegenerative
disorders, such as Alzheimer's or Parkinson's Disease (e.g., again,
by targeting the delivery of neuroprotective substances or growth
factors, or by targeting the delivery of other neuroprotective
factors such as amyloid precursor proteins or protease
nexin-1).
[0034] As discussed in the ensuing Examples, and with reference to
the second therapeutic mechanism indicated above, the neural
progenitor cells of the present invention are also able to replace
neurons and glia in vivo. The cells may therefore be used to
replace diseased or damaged neural tissue, and, owing to the cells'
additional capacity to track diseased or damaged tissue in vivo,
once administered, the cells may configure themselves to an
appropriate physiological site to effect this therapeutic
mechanism.
[0035] Given the ability of the neural progenitor cells of the
present invention to replace lost or damaged neural tissue
function, these cells may be useful in the treatment of numerous
neuropathological conditions, many of which are similar to those
enumerated above. By way of example, even in a state where the
cells have not been transfected or otherwise caused to carry a
particular gene, the cells may be used in the treatment of brain
tumors, brain ischemia, spinal cord injury, and various
neurodegenerative disorders.
[0036] Neural progenitor cells that are, in fact, transfected or
otherwise caused to carry a desirable gene may also provide the
additional neural cell function replacement capacity discussed in
this mechanism; thereby imparting a dual treatment effect to the
recipient. The dual treatment effect may include the replacement of
lost or damaged cell function (e.g., as per the second therapeutic
mechanism) in conjunction with the targeted delivery of a
beneficial compound to that same region (e.g., as per the first
therapeutic mechanism). Therefore, in the illustrative instance of
brain tumor treatment, the neural progenitor cells may be
transfected with a gene that induces the secretion of cytokines
(e.g., tumor necrosis factor (TNF) or interleukin-1 (IL-1)), and
implanted or otherwise administered to the brain of a recipient.
Once administered, the cells may track the tissue damaged by the
tumor, replacing at least a portion of the lost brain function,
while simultaneously secreting cytokines that may induce an immune
response against the tumor cells. This dual treatment effect is
further described in the ensuing Examples.
[0037] Neural progenitor cells developed through culture as
described above may be implanted in or otherwise administered to a
mammal to effect the therapeutic mechanisms previously discussed.
Once implanted or otherwise administered, these cells may relocate
to an area of diseased tissue, such as, but not limited to, brain
tumors, tissue damaged by stroke or other neurodegenerative
disease, and the like. Moreover, the neural progenitor cells may
multiply in vivo, and may further follow diseased tissue as it
spreads (e.g., as a tumor spreads). Implantation may be performed
by any suitable method as will be readily ascertained without undue
experimentation by one of ordinary skill in the art, such as
injection, inoculation, infusion, direct surgical delivery, or any
combination thereof.
EXAMPLES
[0038] All references cited herein are hereby incorporated by
reference in their entirety. The following examples are typical of
the procedures that may be used to culture neural progenitor cells
according to a method of the present invention. Further examples
are typical of the procedures that may be used to perform gene
transfer into these cells and/or implant these cells into a patient
to treat a neurological disorder in accordance with another
embodiment of the present invention. Modifications of these
examples will be apparent to those skilled in the art.
Example 1
Isolation and Preparation of Neural Progenitor Cells
[0039] Whole bone marrow was harvested from the femurs of adult
Fisher rats between 16 and 24 weeks of age. Cultures were plated on
poly-D-lysine coated 24 well plates at a density of 106 cells per
well. The cells were cultured in serum-free Dulbecco's modified
Eagle medium (DMEM)/F-12 medium supplemented with B27 (obtained
from Gibco BRL; Gaithersburg, Md.), 20ng/ml FGF-2 and 20 ng/ml EGF
(both available from Sigma Chemical Co.; St. Louis, Mo.;
hereinafter "Sigma"), along with penicillin and streptomycin (both
available from Omega Scientific, Inc.; Tarzana, Calif.).
[0040] After four days in culture, numerous floating spheres of
between about 10 to about 100 cells were distinctly visible
separate from an underlying adherent monolayer (FIG. 1A). These
spheres were collected and sub-cultured separately (FIG. 1B). The
cellular aggregates continued to expand and the rate of
proliferation remained stable even after multiple disassociations
and passages. Numerous cells in these spheres tested positive for
nestin (FIGS. 2C and 2D), a known marker for neural stem cells. U.
Lendahl et al., Cell 60:585-595 (1990).
[0041] Spheres taken after four days of sub-culture were plated
onto laminin-coated 24 well plates in media devoid of growth
factors. The spheres attached and cells at the outer margins of
each sphere began to develop processes and migrate away from the
primary site of attachment (FIG. 1C). Formed spheres detached from
the bottom of the plates and thereafter remained free-floating
(FIG. 1D); displaying multiple morphologies (FIG. 4A). Neural
progenitor cells expressed GFAP (FIGS. 4B and 4C) and the early
neuronal marker NeuN (FIGS. 4D and 4E). Scattered cells also
expressed the later neuronal marker MAP2 (FIG. 4F). Bone
marrow-derived spheres were transplanted into the hippocampus of a
syngeneic animal, and cells expressing NeuN were found (FIG. 4G);
some of these cells intagrating into the hippocampal structure
(FIG. 4H). Data was also collected utilizing alternate antibodies
for immunocytochemistry, including CNPase (FIG. 4I) and NF200
(FIGS. 4J and 4K).
[0042] Neurospheres (i.e., cells derived from neural tissue) and
bone marrow-derived spheres were morphologically indistinguishable
(FIGS. 2A and 2B). The pattern of nestin expression was similar in
both (FIGS. 2C and 2D); although bone marrow-derived spheres
expressed the ectodermal marker vimentin (FIG. 3A) and also
displayed a weak staining for fibronectin (FIG. 3B). Furthermore,
the bone marrow-derived spheres exhibited strong expression of CD90
(FIG. 3C), and the majority of cells in spheres also displayed
nuclear expression of Neurogenin 1 (FIG. 3D).
Example 2
Gene Transfer Into Neural Progenitor Cells Utilizing
Replication-Deficient Adenoviral Vectors
[0043] Type 5 replication-deficient adenoviral vectors bearing
either the reporter gene for P-galactosidase or the gene for the
cytokine IL-12 were used to infect neural progenitor cells in
vitro. 24 hours following infection, successful gene transfer was
confirmed using X-gal staining (X-gal Staining Assay Kit available
from Gene Therapy Systems, Inc.; San Diego, Calif.) for
.beta.-galactosidase-bearing adenoviral-infected progenitor cells,
and an IL-12 Enzyme Linked Immunosorbent Assay ("ELISA" kit
available from BD Pharmingen; San Diego, Calif.) for IL-12
gene-bearing adenovirus-infected progenitor cells.
[0044] Successful gene transfer of .beta.-galactosidase was
confirmed by positive staining for the X-gal and
.beta.-galactosidase-generated blue precipitate in the
.beta.-galactosidase-bearing adenovirus-infected progenitor cells
(FIG. 5). Successful gene transfer of IL-12 was confirmed by the
positive photochromic ELISA reaction in media harvested from the
IL-12 gene-bearing adenovirus-infected progenitor cells (Table 1).
TABLE-US-00001 TABLE 1 Detection of IL-12 Secretion by ELISA IL-12
Detection by ELISA Cells infected in vitro with AdIL-12 >>2
ng/ml Cells infected in vitro with AdLacZ 4 pg/ml Mock infected
cells Not detected
Example 3
Gene Transfer Into Neural Progenitor Cells Utilizing a
Double-Mutated Herpes Simplex Virus Type I
[0045] A herpes simplex type I virus deleted for the genes encoding
the latency activated transcript (LAT) and gamma 34.5 genes (virus
denoted DM33) was utilized. The virus contained the gene for GFP
under the control of the powerful LAT promoter, and was therefore
able to confer constitutive expression of GFP into any successfully
infected cell. This vector was used to infect neural progenitor
cells in vitro. 72 hours after infection, successful gene transfer
was confirmed by viewing GFP expression under a fluorescent light
microscope (FIG. 6).
[0046] GFP expression was visible in neural progenitor cells 72
hours following infection with DM33. This confirmed the ability to
successfully utilize herpes simplex type I for gene transfer to
neural progenitor cells.
Example 4
Neural Progenitor Cells are Capable of Differentiating into
Astrocytes, Neurons, and Oligodendroglia
[0047] Neural progenitor cells were replated in vitro in culture
media devoid of essential growth factors and supplemented with
retinoic acid (a known stimulator of differentiation). Culture
surfaces were coated with poly-D-lysine (available from Sigma) to
promote attachment of differentiating cells.
[0048] After three to four days, neural progenitor cells had
attached to the culture surface and differentiated into astrocytes,
neurons, and oligodendroglia. The presence of these cells was
specifically confirmed by positive immunocytochemical staining
populations for known markers of all three lineages. Specifically,
astrocytes in the culture population were positive for GFAP;
neurons were positive for .beta.-III tubulin; and oligodendroglia
were positive for CNPase. This confirms the multipotency and true
progenitor nature of the neural progenitor cells of the present
invention.
Example 5
[0049] Neural Progenitor Cells Track Spreading Brain Tumor Cells In
Vivo
[0050] Neural progenitor cells were infected with
replication-deficient adenovirus bearing the gene for
.beta.-galactosidase as described in Example 2 above. These cells
were then transplanted intratumorally into C57bl/6 mice bearing
established GL26 brain tumors in their right cerebral hemispheres,
respectively. After eleven days, the mice were euthanized, and
their brains were immediately harvested, frozen, and sectioned
using a cryostat (available from Janis Research Company, Inc.;
Wilmington, Mass.). The frozen sections were then stained using an
X-gal staining solution to detect the presence of
.beta.-galactosidase-expressing neural progenitor cells within the
brain tumors.
[0051] Neural progenitor cells were clearly visible within the main
tumor mass. In addition, neural progenitor cells could clearly be
seen tracking pockets of tumor cells that were migrating away from
the main tumor mass. This clearly demonstrated the ability of
neural progenitor cells to actively follow pockets of tumor cells
that disseminate through the brain.
Example 6
Neural Progenitor Cells Track Ischemic Brain Injury In Vivo
[0052] The middle cerebral artery of Wistar rats was occluded with
a thread embolus for two hours. A drop in perfusion pressure
verified effectiveness of occlusion. Neural progenitor cells were
infected with replication-deficient adenovirus bearing the gene for
.beta.-galactosidase as described in Example 2 above. The cells
were infused intracranially either immediately or two hours
following middle cerebral artery occlusion. After 48 hours, the
rats were euthanized, and their brains were immediately harvested,
frozen, and sectioned. The fresh frozen sections were then stained
using an X-gal staining solution to detect the presence of
.beta.-galactosidase-expressing neural progenitor cells in the
brain.
[0053] Neural progenitor cells were clearly identifiable in the
sectioned brains indicating that these cells can readily cross the
blood brain barrier. The transplanted cells were distributed
throughout the ischemic part of the brain, mostly as single cells
infiltrating the pathological tissue (FIG. 8). While not wishing to
be bound by any theory, it is believed that this may be part of the
cells' response to chemotactic stimuli originating from the damaged
tissue. The cells could also be found in normal parts of the brain
and some cells were located in the meninges.
Example 7
Neural Progenitor Cells May Be Generated From Fetal Brain
Tissue
[0054] Neurospheres were generated from primary fetal brain
culture, in a manner similar to that described with respect to bone
marrow in Example 1, above (i.e., cells were cultured in serum-free
DMEM/F-12 medium supplemented with B27, 20 ng/ml FGF-2 and 20 ng/ml
EGF, along with penicillin and streptomycin). Neural stem cells
grew into spherical aggregates 2-3 days following harvest and
cultured in growth factor- supplemented media (FIG. 7A). These
neurospheres were comprised of neural progenitor cells expressing
nestin (FIG. 7B).
[0055] The neural stem cells were re-plated in modified culture
conditions, after the cells were induced to differentiate. Neurons
expressed .beta.-III tubulin (FIG. 7C), astrocytes expressed GFAP
(FIG. 7D), and oligodendrocytes expressed CNPase (FIG. 7E). Neural
stem cells infected in vitro with AdLacZ expressed
.beta.-galactosidase (FIG. 7F).
Example 8
Neural Progenitor Cells Track Tumor Cells In Vivo
[0056] Tumors from glioma-bearing mice inoculated with neural
progenitor cell-LacZ were stained with X-gal and counter-stained
with Neutral red. Four distinct patterns of tumor spread were
detected and neural progenitor cells were found tracking migrating
glioma in each case: (1) a thin outgrowth of tumor cells deep into
adjacent normal brain; (2) a direct extension of tumor mass into
adjacent tissue; (3) a migration of glioma cells away from the
primary tumor bed along a white matter tract; and (4) a tumor
microsatellite independent of a main tumor mass (FIGS. 9A-9D,
respectively). Interspersed with the tumor cells depicted in the
tumor microsatellite (FIG. 9D) were .beta.-galactosidase positive
neural progenitor cells; revealed with a high power photomicrograph
(FIG. 9E).
[0057] Neural progenitor cells were inoculated into a cerebral
hemisphere contralateral to an existing tumor. The progenitor cells
were introduced into the left cerebral hemisphere (FIG. 9F, left
panel), but demonstrated specific, non-random migration into the
vicinity of the tumor in the opposite hemisphere (FIG. 9F, right
panel and inset box). Neural progenitor cells appear blue
(indicating expression of .beta.-galactosidase), whereas tumor
cells appear red (hypercellular areas were stained intensively with
neural red). Thus, neural progenitor cells display strong tropism
for disseminating glioma in vivo.
Example 9
Neural Progenitor Cells Transfected With Cytokines Induce Localized
Immune Response In Vivo
[0058] Neural progenitor cells were transfected with genes that
induced them to secrete either IL-12, 3T3-IL-12 or LacZ, as
described in Example 2, above. These neural progenitor cells were
inoculated into the glioma-bearing brains of rats.
[0059] A flow cytometry analysis indicated robust intratumoral
T-ell infiltration in brains inoculated with neural progenitor
cells secreting IL-12 and 3T3-IL-12 (FIG. 10A, left and center
panels, respectively). However, intratumoral T-cell content of
brains inoculated with neural progenitor cells secreting LacZ was
much lower (FIG. 10A, right panel) and was comparable to
infiltration seen in mock-transfected neural progenitor cells and
saline-inoculated gliomas (data not shown).
[0060] Tumors treated with neural progenitor cells secreting IL-12
demonstrated robust CD4+ and CD8+T-cell infiltration (FIG. 10D,
left and right panels, respectively), with numerous aggregates
along the tumor/normal tissue boundary (FIG. 10D). Tumors treated
with neural progenitor cells secreting 3T3-IL-12 also demonstrated
CD4+ and CD8+T-cell infiltration (FIG. 10B, left and right panels,
respectively), with positive cells interspersed in tumor tissue.
However, tumors treated with neural progenitor cells secreting LacZ
displayed negligible infiltration of tumors by CD4+ or CD8+T-cells
(FIG. 10C, left and right panels, respectively).
[0061] In a comparative analysis of T-cell infiltration in
comparable outgrowths from a primary tumor bed, tumor
microsatellites in brains treated with neural progenitor cells
secreting IL-12 demonstrated robust T-cell infiltration, whereas
those in brains treated with neural progenitor cells secreting
3T3-IL-12 did not (FIG. 10E, left and right panels,
respectively).
Example 10
Neural Progenitor Cells Transfected With .beta.-Galactosidase Track
Tumor Cells In Vivo
[0062] RG2 tumor cells (100,000 in 5 ul of media) were
stereotactically implanted into the striatum of Wistar rats. Two
days following tumor implantation, 30,000 bone marrow derived cells
infected with adenovirus carrying the .beta.-galactosidase gene
were implanted into the same site. The immunohistological analysis
was done 60 days following cell implantation. As depicted in FIG.
12, the cells, stained for LacZ, can be seen in the tumor outgrowth
migrating out from the main tumor mass at 10.times. (FIG. 12A) and
40.times. (FIG. 12B) magnification. The sections were
counterstained with hematoxylin.
[0063] While the description above refers to particular embodiments
of the present invention, it will be understood that many
modifications may be made without departing from the spirit
thereof. The accompanying claims are intended to cover such
modifications as would fall within the true scope and spirit of the
present invention. The presently disclosed embodiments are
therefore to be considered in all respects as illustrative and not
restrictive, the scope of the invention being indicated by the
appended claims, rather than the foregoing description, and all
changes that come within the meaning and range of equivalency of
the claims are therefore intended to be embraced therein.
* * * * *