U.S. patent application number 11/340322 was filed with the patent office on 2006-06-22 for use of marrow-derived glial progenitor cells as gene delivery vehicles into the central nervous system.
This patent application is currently assigned to The Government of the United States of America as represented by the Secretary of the Dept. of Healt. Invention is credited to Martin A. Eglitis, Eva Mezey, Mary Maral Mouradian.
Application Number | 20060134081 11/340322 |
Document ID | / |
Family ID | 27365063 |
Filed Date | 2006-06-22 |
United States Patent
Application |
20060134081 |
Kind Code |
A1 |
Eglitis; Martin A. ; et
al. |
June 22, 2006 |
Use of marrow-derived glial progenitor cells as gene delivery
vehicles into the central nervous system
Abstract
The present disclosure relates to a method for introducing a
hematopoietic cell into the brain of a mammal, by administering
bone marrow-derived progenitor cells into the body of the mammal by
intravenous injection. The bone marrow-derived cell is preferably a
cell that differentiates into a glial cell. The disclosure also
relates to a method for delivery of therapeutic protein molecules
into the brain of a mammal, by administering to a mammal an
effective amount of bone marrow-derived progenitor cells which
contain a gene having a nucleic acid sequence that encodes a
functional therapeutic protein. Isolated recombinant cells and a
pharmaceutical composition are also provided.
Inventors: |
Eglitis; Martin A.;
(Indianapolis, IN) ; Mezey; Eva; (Rockville,
MD) ; Mouradian; Mary Maral; (Bethesda, MD) |
Correspondence
Address: |
KLARQUIST SPARKMAN, LLP
121 S.W. SALMON STREET
SUITE #1600
PORTLAND
OR
97204-2988
US
|
Assignee: |
The Government of the United States
of America as represented by the Secretary of the Dept. of
Healt
|
Family ID: |
27365063 |
Appl. No.: |
11/340322 |
Filed: |
January 25, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10122703 |
Apr 11, 2002 |
7022321 |
|
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11340322 |
Jan 25, 2006 |
|
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09819096 |
Feb 16, 2001 |
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10122703 |
Apr 11, 2002 |
|
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09058160 |
Apr 10, 1998 |
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09819096 |
Feb 16, 2001 |
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60036592 |
Apr 10, 1997 |
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|
Current U.S.
Class: |
424/93.21 ;
435/372; 435/456 |
Current CPC
Class: |
C12N 2510/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 48/00 20130101;
A61K 35/28 20130101; C12N 2501/23 20130101; A61K 38/185 20130101;
C12N 2510/02 20130101; C07K 14/475 20130101; C12N 2501/125
20130101; A61K 38/185 20130101; C12N 5/0647 20130101; A61K 35/28
20130101; C12N 15/87 20130101; C12N 2799/027 20130101 |
Class at
Publication: |
424/093.21 ;
435/456; 435/372 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 5/08 20060101 C12N005/08; C12N 15/867 20060101
C12N015/867 |
Claims
1. A method of treating ischemia in the brain of a subject,
comprising: administering harvested bone marrow cells to a subject;
and allowing the bone marrow cells to migrate to an ischemic region
in the brain of the subject, thereby treating the ischemic region
in the brain of the subject.
2. The method of claim 1, wherein the bone marrow cells are
transfected with a glial cell line-derived neurotrophic factor
(GDNF) gene.
3. The method of claim 1, wherein the bone marrow cells
differentiate into astroglia and microglia in the brain of the
subject.
4. The method of claim 1, wherein the bone marrow cells migrate to
the cortex, hippocampus, thalamus, brainstem or cerebellum of the
brain.
5. The method of claim 2, wherein the harvested bone marrow cells
are transfected with a vector comprising the GDNF gene.
6. The method of claim 5, wherein the vector is a retroviral
vector.
7. The method of claim 6, wherein the retroviral vector is a
Moloney murine leukemia virus vector.
8. The method of claim 1, further comprising culturing the
harvested bone marrow in vitro in a cell culture medium comprising
IL-3, IL-6, and stem cell factor prior to the administering
step.
9. The method of claim 1, wherein the subject has been sub-lethally
irradiated prior to administration of the harvested bone marrow
cells.
10. A method of treating cortical ischemia, comprising: culturing
harvested bone marrow cells in a cell culture medium comprising
IL-3, IL-6, and stem cell factor; transfecting the harvested bone
marrow cells with a Moloney murine leukemia virus vector comprising
a gene for glial cell line-derived neurotrophic factor (GDNF);
administering the transfected cells intravenously to a subject
having cortical ischemia; and allowing the transfected cells to
migrate to the brain of the subject and express the GDNF gene,
thereby treating the cortical ischemia.
11. A method of increasing glial cells in the brain of a subject,
comprising: administering harvested bone marrow cells to a subject;
and allowing the bone marrow cells to migrate to the brain of a
subject and differentiate into glial cells, thereby increasing
glial cells in the brain of the subject.
12. The method of claim 11, wherein the glial cells are astroglia
and/or microglia.
13. The method of claim 11, wherein the bone marrow cells migrate
to the cortex, hippocampus, thalamus, brainstem, cerebellum, or an
ischemic region of the brain.
14. The method of claim 11, further comprising culturing the
harvested bone marrow in vitro in a cell culture medium comprising
IL-3, IL-6, and stem cell factor prior to the administering
step.
15. The method of claim 11, wherein the subject has been
sub-lethally irradiated prior to administration of the harvested
bone marrow cells.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional of U.S. patent application Ser. No.
10/122,703, filed Apr. 11, 2002, now allowed, which is a
continuation-in-part of U.S. patent application Ser. No.
09/819,096, filed Feb. 16, 2001, now abandoned, which is a
continuation of U.S. patent application Ser. No. 09/058,160, filed
Apr. 10, 1998, now abandoned, which claims the benefit of U.S.
Provisional Application No. 60/036,592, filed Apr. 10, 1997.
FIELD
[0002] The present disclosure relates to methods for introducing
hematopoietic cells into the brain of a mammal, the differentiation
of adult bone marrow cells into glial cells, and the use of
marrow-derived glial progenitor cells as gene delivery vehicles
into the brain.
BACKGROUND
[0003] Glial cells are thought to derive embryologically from
either myeloid cells of the hematopoietic system (microglia) or
neuroepithelial progenitor cells (astroglia and oligodendrocytes).
However, it is unclear whether the glia in adult brains free of
disease or injury originate solely from cells present in the brain
since the fetal stage of development, or if there is further input
into such adult brains from cells originating outside the central
nervous system (CNS).
[0004] Besides the cells of the vasculature, the brain comprises
two general cell types: neurons and glial cells. Glial cells
provide physiological support to neurons and repair neuronal damage
due to injury or disease. Macroglia (astroglia and oligodendroglia)
are generally considered to be derived from neuro-ectoderm and are
believed to be developmentally distinct from microglia (1).
However, the developmental origin of microglia remains debatable
(2,3). The two major views are that they derive either from
neuro-epithelial cells (4-6) or from hematopoietic cells (i.e.,
monocytes) (7, 8). The extent to which cells outside the CNS
contribute to the maintenance of microglia in adults remains
debatable (compare (9) and (10)), and no such contribution to adult
neurons or macroglia has been previously described.
SUMMARY OF THE DISCLOSURE
[0005] Heretofore, gene therapy in the brain relied upon surgically
implanting the transfected cells into the recipient brain. It was
unknown prior to our disclosure that cells of the hematopoietic
system are a source of progenitor cells for the CNS, such that
these cells can be used as a gene therapy delivery vehicle into the
brain.
[0006] We tested the ability of hematopoietic cells to contribute
to the CNS, by transplanting adult female mice with donor bone
marrow cells genetically marked either with a retroviral tag or by
using male donor cells. We monitored the appearance of the cells in
the brain using in situ hybridization histochemistry (ISHH)
combined with immunohistochemistry. We also performed double ISHH
with digoxigenin and radioactively labeled probes to analyze which
cell types might be derived from bone marrow stem cells. We
detected a continuing influx of hematopoietic cells into the brain.
Marrow-derived cells were already detected in the brains of mice
three days after transplant and their numbers increased over the
next several weeks, exceeding 14,000 cells per brain in several
animals. Marrow-derived cells were widely distributed throughout
the brain, including the cortex, hippocampus, thalamus, brainstem,
and cerebellum. When ISHH was combined with immunohistochemical
staining using lineage-specific markers, some bone marrow-derived
cells were positive for the microglial antigenic marker F4/80.
Other marrow-derived cells surprisingly expressed the astroglial
marker glial fibrillary acidic protein (GFAP). These results
indicate that some microglia and astroglia arise from a precursor
that is a normal constituent of adult bone marrow.
[0007] The results reported here confirm that cells derived from
the bone marrow can migrate into the brains of adult mice.
Furthermore, we have found that this migration is rapid, with
numerous cells present by the third day after transplant. These new
cells are distributed throughout the brain, and appear to reside
within the parenchyma, since perfusion with PBS does not remove
them. Occasional donor marrow-derived cells were found in
association with vascular structures. Moreover, densities of donor
cells in the parenchyma paralleled the capillary density of a given
region. For instance, cortex, with fewer capillaries, had a lower
cell density than the more vascularized choroid plexus. Regions
with a higher capillary density, such as the area postrema, also
had the highest density of marrow-derived cells within the
parenchyma.
[0008] Double-labeling analyses show that at least some bone
marrow-derived cells acquire microglial antigenic markers. However,
we also observed many cells positively labeled by ISHH that did not
express the F4/80 antigen. This may be due simply to a level of
antigen below the limits of detection in our assay.
[0009] Alternatively, it is possible that the F4/80 marker is
expressed on marrow-derived cells only after they fully
differentiate into microglia, while less mature microglial
precursors are not recognized by the antibody to F4/80.
Nonetheless, our results strongly support the view that
hematopoietic cells outside the CNS contribute to the maintenance
of microglia in healthy adults. While a partial CNS origin of adult
microglia cannot be excluded, our data is inconsistent with an
exclusively CNS origin. Moreover, although our experiments did not
examine fetal origins of microglia, the finding of
hematopoietically-derived microglia in healthy adults is also
consistent with a hematopoietic origin of microglia in
development.
[0010] Surprisingly, we found that some hematopoietic cells (tagged
either with a retroviral vector or by transplant of male cells into
a female recipient) give rise to cells other than microglia,
specifically to cells that exhibit astroglial markers. Although
this observation is unexpected, it is based on identical results in
multiple animals using two independent means of cell tagging with
both cytoplasmic and nuclear markers.
[0011] The appearance of marrow-derived astroglia seems a normal
process in these animals. Because the number of marrow-derived
cells detected in the brain increased over time, their appearance
does not appear to be a consequence of the transplantation
procedure itself. If appearance in the brain was a by-product of
transplantation, one would expect tagged cell numbers in the brain
to peak and then decline, which was not observed. Rather, the data
is consistent with existence of cells, amongst the populations of
marrow-engrafting cells, capable of continuous generation of
progenitors that migrated to the brain. Interestingly, cells with
marrow markers were seen in the ventricular ependyma. In fact, in
many animals, marrow-derived cells could be found concentrated
sub-ependymally (Mezey & Eglitis, in preparation). The
subependymal zone is an important source of neuronal and glial
progenitors during development (24) and in adults (27). Finding
bone-marrow derived cells in this location opens the possibility
that such cells receive cues guiding their differentiation once
they enter the brain. Studies evaluating this possibility are
ongoing.
[0012] No obvious pathology such as gliosis was detected in the
brain of any transplant recipient (n=46). Some recipient animals
were irradiated before receiving bone marrow transplants to see if
marrow purging enhanced engraftment and seeding of implanted cells.
However, radiation dosages were at least one order of magnitude
below those known to induce pathological changes in the CNS (29).
Indeed, we found preconditioning of recipients was not necessary.
Male donor cells engrafted and persisted for at least 10 weeks even
without irradiation. Furthermore, as many Y chromosome/GFAP double
positive cells were seen with as without irradiation. The wide
distribution of GFAP-positive cells in both gray and white matter
demonstrates that bone marrow-derived progenitors are not
restricted to differentiate into a particular subclass of
astroglia. That is, marrow-marked cells contributed to both fibrous
astrocytes in the white matter and protoplasmic astrocytes in the
gray matter.
[0013] One alternative explanation for our observing GFAP staining
of cells bearing marrow markers is that processes from endogenous
astroglia surround the in-migrating cells from the donor marrow.
However, some of our data argue against this possibility. First,
cytoplasmic neo.sup.R ISHH labeling coincided with cytoplasmic GFAP
immunostaining. Furthermore, upon evaluation of fifty to 100 male
nuclei associated with GFAP staining, no nuclei were seen that
could be considered part of an engulfing astroglial cell. If
endogenous astroglia were the source of the GFAP staining
associated with donor male nuclei, one would expect the geometry in
12.mu. sections to reveal the cell body and nucleus corresponding
to the putative engulfing processes in at least a few cases. After
analyzing dozens of sections, no such cases were observed.
[0014] Because only about 10% of marrow-derived cells in the brain
exhibit expression of either the microglial F4/80 antigen or the
astroglial marker GFAP, the identity of the majority of bone
marrow-derived cells remains an open question. Nonetheless, there
is clearly a measurable contribution by cells of hematopoietic
origin to the glial cell population of the brain in adult mice,
which indicates that some glial progenitors reside outside the CNS.
The observation of marrow-derived astroglia in the optic tract
demonstrates that some of these marrow-derived progenitors may be
similar to the previously recognized astroglial precursor (30).
[0015] Microglia and astroglia respond differently to brain injury.
In fact, astrogliosis often appears to be a response to primary
microgliosis (31, 32). There is also evidence that different brain
lesions elicit different microglial and astroglial responses (33).
Our results provide a way that gene transfer into hematopoietic
progenitors can be used to introduce genes into microglia and
astroglia that then would participate in the gliosis associated
with a CNS pathology. The detection of marrow-derived cells in
brains within days of transplantation provides a method in which
genetically altered hematopoietic cells could be used to treat
acute diseases of the brain.
[0016] Although many neurotrophic factors show promise in the
treatment of CNS disorders, their use has been hindered by their
inability to cross the blood-brain barrier and by their limited
diffusion into CNS tissues (34). In addition, adverse effects have
been reported after systemic administration of some neurotrophins
(35). Using marrow-derived cells to deliver therapeutic proteins
directly to the site of CNS pathology likely would be more benign
than systemic administration of toxic molecules. In addition, using
vectors with cell type-specific promoters could restrict gene
expression specifically to reactive astroglia or microglia, thereby
providing greater therapeutic precision for gene therapy of CNS
disease.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1 shows the behavior of marrow-derived glia in the rat
ischemic injury model described in Experiment 9. Shown is the
number of marrow-derived cells detected in the lesioned ischemic
vs. the contralateral non-ischemic side of three rat cortexes
following MCA occlusion using Y chromosome-specific
hybridization.
[0018] FIG. 2 shows the results of Experiment 9, in that in each of
the three lesioned animals, more male donor marrow-derived
astrocytes were detected on the ischemic side than on the
non-ischemic side.
[0019] FIG. 3 shows the effect of transplantation with genetically
engineered marrow cells in a mouse model of Parkinson's disease
described in Experiment 10. FIG. 3A is a graph of horizontal
activity versus time. FIG. 3B is a graph of the number of movements
versus time.
DETAILED DESCRIPTION
EXPERIMENT 1
Gene Transfer and Bone Marrow Transplantation
[0020] Gene transfer into hematopoietic precursors was done as
previously described (11, 12), with the addition of stem cell
factor to optimize transduction of reconstituting hematopoietic
stem cells (13). C57BL/6J mice (Jackson Laboratories, Bar Harbor,
Me.), 6-8 weeks old, were used as donors. Forty-eight hours before
marrow harvest, the mice were injected with 5-fluorouracil at a
dose of 150 mg/kg to ablate mature blood cells and thereby induce
progenitor cells into cycle. Upon harvest, marrow was placed into
liquid culture in suspension dishes and grown in Dulbecco's
modified Eagle's medium containing 15% fetal bovine serum
(Whittaker Bioproducts, Walkersville, Md.) and supplemented with
IL-3 (50 ng/ml), IL-6 (100 ng/ml) and stem cell factor (100 ng/ml).
Growth factors were used to maintain early hematopoietic cells in
cycle (13). All were obtained from R & D Systems (Minneapolis,
Minn.). After 48 hr in culture with growth factors, marrow cells
were collected and added to tissue culture dishes containing the
F5B producer cell line at subconfluent density. F5B cells shed the
N2 retroviral vector, packaged with the ecotropic envelope and
carrying the bacterial gene for neomycin resistance (neo.sup.R)
(14). Following 48 hr co-culture with F5B cells, bone marrow cells
were collected by gentle aspiration, suspended to 1.times.10.sup.7
cells/ml in phosphate buffered saline (PBS, in all cases 0.1 M
phosphate, 140 mM NaCl, pH 7.6) and injected intravenously
(2-3.times.10.sup.6 cells/mouse) via the tail vein into
sub-lethally irradiated (4.5 Gy) female
WBB6F1/J-Kit.sup.W/Kit.sup.W-v mice. WBB6F1/J-Kit.sup.W/Kit.sup.W-v
mice are particularly good recipients for bone marrow
transplantation because they have genetically defective stem cells
(15). This gives normal C57BL/6J donor stem cells a strong
repopulating advantage.
[0021] In transplants of male donor marrow into female recipients,
some marrow was marked with retroviral vector as described. In
other cases, marrow was harvested, washed with PBS, and
transplanted directly into recipient mice without culturing in
growth factor-containing medium or irradiation of recipient
animals.
[0022] A total of 46 mice were transplanted, 38 with vector tagged
marrow and 8 with male marrow. Five of the transplants with vector
tagged marrow used male donor cells. Mice were sacrificed at
various times after transplantation. At least two animals were
analyzed at each time point, although more were used at the 14 day
(n=10), 35 day (n=14), and 70 day (n=6) time points. Tissues were
collected and immediately frozen on dry ice for subsequent
sectioning. Some animals underwent cardiac perfusion with PBS
before tissue harvest. Animals for perfusion were anesthetized with
carbon dioxide, then their chest was opened and PBS was introduced
through a cannula placed in the left ventricle. The left atrium was
incised to allow release of blood. Animals were perfused with 50 ml
of ice cold PBS over a period of 5 min.
EXPERIMENT 2
In situ Hybridization Histochemistry
[0023] Tissues were evaluated with both oligonucleotide and RNA
probes. To detect neo R transcripts, two oligonucleotide probes
were prepared, complementary to the sequence of the neo.sup.R gene
either from nucleotides 222-269 or from nucleotides 447-494
(numbering with the A of the initiation codon as 1). The
oligonucleotides were labeled using terminal transferase
(Boehringer-Mannheim, Indianapolis, Ind.) and .sup.35S-dATP (New
England Nuclear, Boston, Mass.) as described previously (16). An
RNA probe, complementary to the entire neo.sup.R coding region, was
labeled with .sup.35S-UTP using SP6 polymerase (17). Labeling with
radioactive probes was detected by dipping hybridized sections in
photographic emulsion. Emulsion was exposed for 14 days, then
developed and sections were stained, air dried, and coverslipped
for microscopic examination. To detect male bone marrow cells
transplanted into female recipients, sequences specific to the
donor mouse Y chromosome were detected using a complementary RNA
probe derived from the plasmid pY353/b (18). GFAP gene expression
was detected using an RNA probe complementary to the entire GFAP
coding region. The Y chromosome and GFAP probes were labeled using
digoxigenin-UTP (19), and digoxigenin labeling was developed for
GFAP using alkaline phosphatase as described (19). For detection of
the donor Y chromosome, before overnight hybridization with
digoxigenin-labeled probes at 55.degree. C., the slides were heated
at 90.degree. C. for 10 minutes in hybridization buffer containing
the probes to improve access to nuclear DNA. The
digoxigenin-labeled Y chromosome was visualized using a
modification (Mezey et al., in preparation) of an immuno-staining
amplification method (20), which results in green fluorescein
isothiocyanate (FITC) fluorescence.
[0024] Twelve-micron thick frozen sections were cut in a cryostat
and ISHH was performed as described previously (16, 17). The
sections were fixed, dehydrated, and delipidated in ethanol and
chloroform and then hybridization buffer containing the probe(s)
was put on the sections. Slides were incubated overnight in a
humidified chamber at 37.degree. C. (for oligonucleotide probes) or
55.degree. C. (for riboprobes).
EXPERIMENT 3
Nuclear Staining
[0025] To confirm that Y chromosome ISHH coincided with cell
nuclei, sections were counterstained with ethidium bromide or
4',6-diamidino-2-phenylindole (DAPI). Staining was detected by
illumination with a mercury lamp using a microscope equipped for
fluorescence micrography.
EXPERIMENT 4
Immunohistochemical Analysis
[0026] For combined ISHH/immunohistochemical analysis, sections
were fixed as described previously (21). They were then incubated
for 30 minutes at room temperature in 3% normal goat serum diluted
in PBS (containing 0.6% Triton-X 100) to block nonspecific binding.
Then, the sections were exposed for one hour at room temperature to
either 1) a polyclonal rabbit antibody that detects the mouse F4/80
monocyte/macrophage marker (22) or 2) a polyclonal rabbit antibody
against the astroglial marker GFAP (Sigma, St. Louis, Mo.) used at
a dilution of 1:2000. Binding of non-labeled primary antisera was
detected with either a biotinylated or goat anti-rabbit IgG
(Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.), both
diluted 1:500. To detect biotinylated secondary antibody, the
sections were incubated for one hour in an avidin-biotin-peroxidase
complex diluted 1:250 in PBS with 0.6% Triton-X 100 (23). The
slides were then transferred into 0.1 M Tris-HCl (pH 7.6) and were
developed using diaminobenzidine as a substrate. Following a
thorough wash, the sections were processed for ISHH. Co-labeling of
cells was determined using a combination of bright-field,
polarized, fluorescent, and epi-illumination microscopy. Controls
for the immunostaining included leaving out the primary antibodies
and using several secondary antibodies (from different species) to
confirm that there was no nonspecific binding.
EXPERIMENT 5
Detection of Donor Cells in the Brain After Bone Marrow
Transplantation
[0027] To evaluate the appearance and distribution of donor cells
in the brains of recipient mice, animals were sacrificed 3, 5, 7,
14, 28, 35, 42 and 70 days after transplantation with bone marrow
cells. At least two animals transplanted with retrovirally tagged
marrow were studied at each time point. Mice transplanted with male
marrow were analyzed at 35 days (n=9) and 70 days (n=4) after
transplantation. Using probes specific to the vector neo R
transcripts, donor cells were detected beginning with day three,
the earliest time of analysis. Many cells were easily detected
throughout the brain by day seven and cells continued to be
detected at all subsequent times. To estimate total number of
neo.sup.R-positive cells in a brain, every 25th section was
collected and all labeled cells in the sections were counted. The
number of labeled cells was multiplied by 25 to arrive at the
approximate total number of marked cells in a brain. These
calculations showed that the overall number of marrow-derived cells
per brain gradually increased with increasing time after
transplantation. Three days after transplant, 500 cells were
detected per brain. Two to 4 weeks after transplant the number of
cells present had increased to at least 2000 per brain. In several
animals more than 10,000 cells per brain were seen, and in one
animal the number of cells was over 30,000.
[0028] At one week, and occasionally at later times, concentrations
of neo.sup.R-marked cells were observed in the basal subarachnoid
space. Bright- and dark-field photographs were taken of the same
section 14 days post-bone marrow transplantation, and cells marked
by the retroviral vector (cells positive by ISHH with
.sup.35S-labeled oligonucleotide or riboprobe) were detected in the
hippocampus, septum, hypothalamus, and within the ependyma of the
third ventricle. Cells were also detected, among other regions, in
the cortex, habenula, pons and cerebellum. Labeled cells were
detected after PBS perfusion, indicating that bone marrow-derived
cells were an integral part of the brain parenchyma. Double
exposures of a brightfield image with a darkfield image were made
of the same area. The darkfield image was photographed using a red
filter so that the autoradiographic grains would appear red.
[0029] Similar regional distribution of donor marrow cells was seen
using the Y chromosome probe to detect male donor cells. Donor
cells (cells positive for the Y chromosome by ISHH) were detected
in several brain regions of a female recipient six weeks after
transplantation with male bone marrow cells. Photomicrographs were
made of a section through the ventral mesencephalon using a
rhodamine filter to excite ethidium bromide staining of the
nucleus, a FITC filter to excite Y chromosome-specific FITC
staining, and/or a double pass filter to show overlap of Y
chromosome labeling and nucleus-specific ethidium bromide
staining.
[0030] Ethidium bromide counter-staining (to highlight the nucleus)
confirmed the nuclear localization of the Y chromosome probe. Many
male donor-derived cells were easily detected throughout the brain
35 days after transplantation and cells continued to be detected at
all subsequent times. Cells positive for the Y chromosome marker
were detected in the mesencephalon, septum, striatum, and habenula.
Cells were also detected in the cortex, pons, and cerebellum, among
other regions (data not shown). Ex vivo manipulation of the bone
marrow cells was not necessary, because male cells were detected in
female recipients' brains even when the transplant was done
immediately after marrow harvest.
[0031] Several parameters were used to verify that the labeling
observed after ISHH was specific. First, no labeling was detected
in any tissues of animals transplanted with non-marked bone marrow
cells. That is, without retroviral tagging, probes for the
neo.sup.R gene exhibited no background labeling, and the Y
chromosome probe did not label female tissues. With the Y
chromosome riboprobe, we also confirmed that both sense and
antisense probes exhibited the same distribution, as expected when
hybridizing to chromosomal DNA. The pattern of retrovirally-labeled
cells was identical in all tissues analyzed, both qualitatively and
quantitatively, regardless of which probe was used. Finally, we
found donor cells in hematologic organs such as bone marrow and
spleen at all time points analyzed. The pattern of engraftment was
qualitatively similar between retrovirally tagged and male donor
cells. However, when female mice were transplanted with
retrovirally tagged male marrow, more donor cells were detected
with the Y chromosome probe than with the neo.sup.R probe. Hence,
not all of the cells migrating from the bone marrow into the brain
expressed the retrovirally introduced neo.sup.R gene at a level
high enough to be detected by ISHH.
EXPERIMENT 6
Labeling of Brain Sections after ISHH with the Microglial Marker
F4/80
[0032] The F4/80 detects the plasma membrane protein F4/80
expressed exclusively on macrophages and microglia (22).
Co-localization in brain sections (cells co-expressing the
microglial marker F4/80 and the neo.sup.R retroviral tag) revealed
cells labeled by the N2 retroviral vector that also expressed the
F4/80 antigen, confirming that bone marrow-derived cells do
contribute to the microglial population in the adult brain.
However, only a small percentage of ISHH-positive cells were
labeled by immunostaining. Similarly, the minority of
antigen-positive cells was doubly labeled by ISHH. The distribution
of doubly labeled cells reflected the distribution of cells labeled
only by ISHH or by immunohistochemistry, i.e., they were widely
distributed throughout the brain.
[0033] The F4/80 monocyte/macrophage antigen was detected by
indirect immunofluorescent antibody labeling, and
.sup.35S-radiolabeled probes were used to hybridize to neo.sup.R
mRNA. Photomicrographs were made of a representative field from an
animal sacrificed 35 days after bone marrow transplantation. In one
representative photomicrograph, a cell in the center stained
positive for the F4/80 antigen (red) and exhibited labeling with
radioactive probe to neo.sup.R transcripts. Darkfield images were
photographed using a green filter so that autoradiographic grains
would appear green (yellow where they overlap red
immunostaining).
EXPERIMENT 7
Labeling of Brain Sections for Both the Astroglial Marker GFAP and
the neo.sup.R Retroviral or Y Chromosome Donor Cell Tag
[0034] The ISHH-positive, F4/80 negative cells could be cells of
the myeloid lineage that had not differentiated to express the
F4/80 antigen. Or, they could represent a contribution of bone
marrow-derived cells to other than myeloid cell lineages. To
distinguish between these alternative possibilities, ISHH-positive
cells were examined for the expression of another lineage marker,
GFAP, specific for astroglia. Surprisingly, we found occasional
cells that were labeled both by ISHH (for the donor marrow
neo.sup.R marker) and by indirect immunohistochemistry (for GFAP).
Photomicrographs were made of cells within the optic tract
expressing GFAP protein using peroxidase-based immunohistochemical
staining combined with ISHH to detect expression of neo.sup.R
transcripts. Double labeled cells were identified adjacent to
clusters of grains indicative of neo.sup.R marked cells that did
not express GFAP and GFAP-positive cells that were not marked with
the retroviral tag. Counting all of the donor cells present in
every 25th section obtained from recipient mice four weeks after
transplantation (n=3), we calculated that as many as
3.times.10.sup.4 neo.sup.R marked donor cells were present per
brain. Of that total donor cell number, we estimated between 0.5%
and 2% exhibited GFAP expression.
[0035] To confirm that GFAP mRNA was present in some
neo.sup.R-positive cells, we also did double ISHH analysis. Cells
co-expressing GFAP and neo.sup.R mRNAs were identified using a
digoxigenin-labeled riboprobe against GFAP mRNA together with a
.sup.35S-labeled probe for the neo.sup.R gene marking the donor
marrow. Photomicrographs were made of sections through the cerebral
cortex. Polarized epifluorescent illumination was used to emphasize
grains indicative of hybridization with .sup.35S-labeled probe for
neo.sup.R. Brightfield illumination was used to emphasize
digoxigenin staining of GFAP transcripts. We found cells labeled
with both probes. Their frequency was approximately equal to the
frequency of the ISHH/GFAP immunostained double cells.
[0036] We also found doubly labeled cells in multiple animals when
ISHH (which was used to detect male cells with the Y chromosome
marker) was combined with immunohistochemistry (to detect GFAP
protein). Using DAPI staining to highlight the nucleus and
three-channel photomicrography, we confirmed that the Y-chromosome
ISHH was associated with the nuclei of GFAP-positive cells.
Photomicrographs were made of double-labeled cells found in the
brains of female recipient mice 10 weeks after bone marrow
transplantation. Male donor cells were detected with a Y
chromosome-specific riboprobe as described above. Astroglia were
identified using a CY3-labeled polyclonal antibody against the
astroglial marker GFAP. In particular, sections were made through
the cortex, through the septum, and through the corpus callosum.
Some sections were illuminated with ultraviolet light to excite
DAPI fluorescent staining of the nucleus. Nuclei from all cells
were stained. Some sections were illuminated to excite FITC
staining of the Y chromosome. Some sections were illuminated to
excite CY3-immunostaining of GFAP.
[0037] Photomicrographs were also made of single fields from
sections through the amygdala. In some photomicrographs, green FITC
staining was used to detect the Y chromosome. In other sections,
red GFAP immunostaining was photographed, while still other
photomicrographs were double exposures of the same field, first
with a double band pass filter to excite FITC and CY3 fluorescence,
then with ultraviolet illumination to excite the blue DAPI
fluorescent staining of the nucleus.
[0038] Dark- and bright-field photographs (of the same section)
were also made that showed several cells exhibiting labeling for
the Y chromosome marker in the fronto/polar cortex of an animal six
weeks after bone marrow transplantation are indicated.
EXPERIMENT 8
Detection of neo.sup.R-Marked Cells in the Brain After Bone Marrow
Transplantation with Retrovirally-Marked Bone Marrow
[0039] Neo.sup.R-marked cells were detected in the brain after bone
marrow transplantation with retrovirally-marked bone marrow.
Photomicrographs were made of representative cells positive for
neo.sup.R transcripts visualized by in situ hybridization
histochemistry (ISHH). Positive cells were found in the region of
the third ventricle of hypothalmus (Hth) of animals sacrificed 35
days after bone marrow transplantation. ISHH-positive cells were
found within the arcuate nucleus of animals sacrificed 14 days
post-bone marrow transplantation. In addition, ISHH-positive cells
were detected in animals 14 days post-bone marrow
transplantation.
[0040] In addition, bright and darkfield photographs were made of
the same field in several of the animals. These photomicrographs
showed cells exhibiting labeling for neo.sup.R transcripts in the
hippocampus of animals two weeks after bone marrow transplantation
(CA3-CA3 region of the hippocampus).
EXPERIMENT 9
Behavior of Marrow-Derived Glia in a Rat Ischemic Injury Model
[0041] Acute cortical ischemia was induced in spontaneously
hypertensive (SHR) rats as follows: Under halothane anesthesia, the
left middle cerebral artery was exposed using a subtemporal
approach (36), and permanently occluded by electrocoagulation
midway between the inferior cerebral vein and lateral olfactory
tract. Forty-eight hours after surgery, animals were sacrificed and
their brains were collected for analysis.
[0042] Y chromosome-specific hybridization indicating
marrow-derived cells in the cortex of brains of transplanted rats
was quantified. The graph in FIG. 1 compares the number of such
cells detected in the lesioned ischemic vs. the contralateral
non-ischemic side of three animals following MCA occlusion. In
addition, the number of marrow-derived cells was compared between
the two hemispheres of two intact animals. Statistical analysis of
10 brain sections from the lesioned animals using the Wilcoxon
Signed Rank test revealed a significantly higher number of Y+
nuclei on the ischemic side compared with the contralateral
non-ischemic cortex (p=0.038). On the other hand, no such
difference was found between the two hemispheres in seven sections
obtained from intact animals (p>0.1).
[0043] The number of Y chromosome-positive astrocytes was
determined in three lesioned and two intact animals. Microscopic
fields were randomly selected based on identifying well-structured
astrocytes using astrocyte-specific anti-GFAP immunofluorescence.
Subsequently, the number of Y chromosome positive astrocytes was
counted, and a comparison made between the two hemispheres. In each
of the three lesioned animals, more male donor marrow-derived
astrocytes were detected on the ischemic side than on the
non-ischemic side (FIG. 2). The increase in number of
marrow-derived astrocytes in lesioned animals was 47% for
experimental animal 1 (E1), 36% for experimental animal 2 (E2), and
14% for experimental animal 3 (E3). In intact rats, generally
smaller differences in the number of marrow derived astrocytes
between the two hemispheres were detected (5% for control 1, 21%
for control 2).
[0044] 1. Summary of Observations in Rats following Middle Cerebral
Artery Occlusion [0045] a. Marrow-derived cells can be detected
throughout the brains of transplanted rats, including in the
ischemic parietal cortex. [0046] b. Marrow-derived astrocytes
participate in the gliosis induced by MCA occlusion. [0047] c. More
marrow-derived cells were detected in the ischemic cortex than in
the contralateral non-ischemic cortex. [0048] d. More
marrow-derived astrocytes were detected in the ischemic cortex than
in the contralateral non-ischemic cortex.
[0049] 2. General Conclusions from Rat Studies
[0050] Marrow-derived cells can be detected throughout the brains
of female rats following transplantation with male bone marrow.
Such cells are detectable in transplant recipients before and after
brain injury. As we previously observed in mice, some
marrow-derived cells differentiate into astrocytes. Such astrocytes
participate in lesion-induced gliosis. Results with the acute MCA
occlusion experiment show that there is some preferential
association of marrow-derived cells, in particular astrocytes, with
the region of gliosis. Preferential association of marrow-derived
glia with regions of gliosis shows that these cells could be used
as vehicles to deliver therapeutic genes to sites of CNS
injury.
EXPERIMENT 10
Effect of Transplantation with Genetically Engineered Marrow Cells
in a Mouse Model of Parkinson's Disease
[0051] Mouse marrow was harvested and transduced with a retroviral
vector as described. Cells were transduced with an MLV-based vector
expressing rat glial cell line-derived neurotrophic factor (GDNF).
This factor has been shown to provide neuroprotective effects in
some rodent models of neurodegeneration. Six weeks after bone
marrow transplantation, mice were treated with
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a substance
which gets metabolized within the brain into a potent neurotoxin
specific for dopaminergic neurons. The significant neuron death in
the substantia nigra resembles that seen in patients with
Parkinson's disease. Using a device to measure the activity level
of mice, the effect of the MPTP treatment on control and
GDNF-transplanted mice was measured.
[0052] In the first 3 days, the overall horizontal activity and
number of movements recorded increased in both the control and
experimental groups (FIG. 3). Between 6 days and 2 weeks after MPTP
treatment, the GDNF-treated group showed a less marked increase in
number of movements than the control group. The level of horizontal
activity also increased less in the mice transplanted with
GDNF-treated marrow than in controls, although the difference
between the groups was less marked than that seen in measurements
of the number of movements. In these preliminary experiments, the
number of animals is too small to assign statistical significance
to the observed differences between control and experimental
groups. However, they warrant the conclusion that marrow-derived
cells migrating into the brain, when engineered to express
neuroprotective growth factors such as GDNF, would protect brains
of treated animals from experimentally induced
neurodegeneration.
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