U.S. patent application number 12/952910 was filed with the patent office on 2011-05-05 for hematopoietic stem cells and methods of treatment of neovascular eye diseases therewith.
This patent application is currently assigned to The Scripps Research Institute. Invention is credited to Karen Da Silva, Martin FRIEDLANDER, Stacey (Hanekamp) Moreno, Atsushi Otani.
Application Number | 20110104131 12/952910 |
Document ID | / |
Family ID | 46205199 |
Filed Date | 2011-05-05 |
United States Patent
Application |
20110104131 |
Kind Code |
A1 |
FRIEDLANDER; Martin ; et
al. |
May 5, 2011 |
HEMATOPOIETIC STEM CELLS AND METHODS OF TREATMENT OF NEOVASCULAR
EYE DISEASES THEREWITH
Abstract
Isolated, mammalian, adult bone marrow-derived, lineage negative
hematopoietic stem cell populations (Lin.sup.- HSCs) contain
endothelial progenitor cells (EPCs) capable of rescuing retinal
blood vessels and neuronal networks in the eye. Preferably at least
about 20% of the cells in the isolated Lin.sup.- HSCs express the
cell surface antigen CD31. The isolated Lin.sup.- HSC populations
are useful for treatment of ocular vascular diseases. In a
preferred embodiment, the Lin.sup.- HSCs are isolated by extracting
bone marrow from an adult mammal; separating a plurality of
monocytes from the bone marrow; labeling the monocytes with
biotin-conjugated lineage panel antibodies to one or more lineage
surface antigens; removing of monocytes that are positive for the
lineage surface antigens from the plurality of monocytes, and
recovering a Lin.sup.- HSC population containing EPCs. Isolated
Lin.sup.- HSCs that have been transfected with therapeutically
useful genes are also provided, and are useful for delivering genes
to the eye for cell-based gene therapy. Methods of preparing
isolated stem cell populations of the invention, and methods of
treating ocular diseases and injury are also described.
Inventors: |
FRIEDLANDER; Martin; (Del
Mar, CA) ; Otani; Atsushi; (Maebashi-City, JP)
; Da Silva; Karen; (Irvine, CA) ; Moreno; Stacey
(Hanekamp); (Spring Valley, CA) |
Assignee: |
The Scripps Research
Institute
|
Family ID: |
46205199 |
Appl. No.: |
12/952910 |
Filed: |
November 23, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10833743 |
Apr 28, 2004 |
7838290 |
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12952910 |
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10628783 |
Jul 25, 2003 |
7153501 |
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10833743 |
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60467051 |
May 2, 2003 |
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60398522 |
Jul 25, 2002 |
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Current U.S.
Class: |
424/93.21 ;
424/93.7; 435/325; 435/372; 435/378 |
Current CPC
Class: |
A61P 9/10 20180101; A61P
27/02 20180101; C12N 5/0692 20130101; A61K 2035/124 20130101; A61K
48/00 20130101; C12N 5/0647 20130101 |
Class at
Publication: |
424/93.21 ;
435/378; 435/372; 435/325; 424/93.7 |
International
Class: |
A61K 35/28 20060101
A61K035/28; C12N 5/0789 20100101 C12N005/0789; A61P 27/02 20060101
A61P027/02 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] A portion of the work described herein was supported by
grant number CA92577 from the National Cancer Institute and by
grants number EY11254, EY12598 and EY125998 from the National
Institutes of Health. The United States Government has certain
rights in this invention.
Claims
1. A method of isolating an adult bone marrow-derived, lineage
negative hematopoietic stem population including endothelial
progenitor cells comprising the steps of: (a) extracting bone
marrow from an adult mammal; (b) separating a plurality of
monocytes from the bone marrow; (c) labeling the monocytes with
biotin-conjugated lineage panel antibodies to one or more lineage
surface antigens selected from the group consisting of CD2, CD3,
CD4, CD11, CD11a, Mac-1, CD14, CD16, CD19, CD24, CD33, CD36, CD38,
CD45, Ly-6G, TER-119, CD45RA, CD56, CD64, CD68, CD86, CD66b,
HLA-DR, and CD235a; (d) removing monocytes that are positive for
said one or more lineage surface antigens from the plurality of
monocytes and recovering a population of lineage negative
hematopoietic stem cells containing endothelial progenitor
cells.
2. The method of claim 1 wherein the mammal is a human.
3. The method of claim 1 wherein the mammal is a human and the
monocytes are labeled in step (c) with biotin-conjugated lineage
panel antibodies to CD2, CD3, CD4, CD11a, Mac-1, CD14, CD16, CD19,
CD33, CD38, CD45RA, CD64, CD68, CD86, and CD235a.
4. The method of claim 2 wherein the mammal is a human and the
method includes the additional steps of labeling the monocytes with
a biotin-conjugated CD133 antibody and recovering a population of
CD133 positive, lineage negative hematopoietic stem cells.
5. The method of claim 2 wherein the mammal is a human and the
method includes the additional steps of labeling the monocytes with
a biotin-conjugated CD133 antibody, removing CD133 positive cells,
and recovering a population of CD133 negative, lineage negative
hematopoietic stem cells.
6. A hematopoietic stem cell population isolated by the method of
claim 1.
7. A method of enhancing retinal neovascularization in a mammal
comprising intravitreally injecting lineage negative hematopoietic
stem cell population of claim 6 into the eye of a mammal in need of
retinal neovascularization wherein the stem cells are derived from
bone marrow of the same species of mammal as the species into whose
eye the cells are injected.
8. The method of claim 7 wherein the stem cells are derived from
the bone marrow of the same individual mammal into whose eye the
stem cells are injected.
9. The method of claim 7 wherein the mammal is a human.
10. A method of treating an ocular disease in a mammal comprising
isolating from the bone marrow of the mammal a lineage negative
hematopoietic stem cell population that includes endothelial
progenitor cells by: (a) extracting bone marrow from a mammal
suffering from an ocular disease; (b) separating a plurality of
monocytes from the bone marrow; (c) labeling the monocytes with
biotin-conjugated lineage panel antibodies to one or more lineage
surface antigens selected from the group consisting of CD2, CD3,
CD4, CD11, CD11a, Mac-1, CD14, CD16, CD19, CD24, CD33, CD36, CD38,
CD45, Ly-6G, TER-119, CD45RA, CD56, CD64, CD68, CD86, CD66b,
HLA-DR, and CD235a; (d) separating monocytes that are positive for
said one or more lineage surface antigens from the plurality of
monocytes and recovering a population of lineage negative
hematopoietic stem cells containing endothelial progenitor cells;
and subsequently intravitreally injecting the isolated stem cells
into an eye of the mammal in a number sufficient to ameliorate the
effects of the disease.
11. The method of claim 10 wherein the number of stem cells is
effective for repairing retinal damage of the mammal's eye.
12. The method of claim 10 wherein the number of stem cells is
effective for stabilizing retinal neovasculature of the mammal's
eye.
13. The method of claim 10 wherein the number of stem cells is
effective for maturing retinal neovasculature of the mammal's
eye.
14. A transfected lineage negative hematopoietic stem cell
population comprising a stem cell population of claim 6 transfected
with a gene that operably encodes a therapeutically useful
peptide.
15. The transfected stem cell population of claim 14 wherein the
therapeutically useful peptide is an anti-angiogenic peptide.
16. The transfected stem cell population of claim 14 wherein the
anti-angiogenic peptide is a protein fragment.
17. The transfected stem cell population of claim 14 wherein the
therapeutically useful peptide is a neurotrophic agent.
18. The transfected stem cell population of claim 17 wherein the
neurotrophic agent is selected form the group consisting of nerve
growth factor, neurotrophin-3, neurotrophin-4, neurotrophin-5,
ciliary neurotrophic factor, retinal pigmented epithelium-derived
neurotrophic factor, insulin-like growth factor, glial cell
line-derived neurotrophic factor, and brain-derived neurotrophic
factor.
19. A method of inhibiting retinal angiogenesis in the eye of a
mammal comprising intravitreally injecting a transfected stem cell
population according to claim 15 into the eye of the mammal.
20. A method of inhibiting retinal neuronal degeneration in the eye
of a mammal comprising intravitreally injecting a transfected stem
cell population according to claim 17 into the eye of the
mammal.
21. A transfected lineage negative hematopoietic stem cell
population prepared by: (a) extracting bone marrow from an adult
mammal; (b) separating a plurality of monocytes from the bone
marrow; (c) labeling the plurality of monocytes with
biotin-conjugated lineage panel antibodies to CD2, CD3, CD4, CD11,
CD11a, Mac-1, CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45,
Ly-6G, TER-119, CD45RA, CD56, CD64, CD68, CD86, CD66b, HLA-DR, and
CD235a; (d) separating monocytes that are positive for said one or
more lineage surface antigens from the plurality of monocytes and
recovering a population of lineage negative hematopoietic stem
cells containing endothelial progenitor cells; and (e) transfecting
the lineage negative hematopoietic stem cells recovered in step (d)
with a polynucleotide that operably encodes a therapeutically
useful peptide.
22. The transfected stem cell population of claim 21 wherein the
therapeutically useful peptide is an anti-angiogenic peptide.
23. The transfected stem cell population of claim 21 wherein the
therapeutically useful peptide is a neurotrophic agent.
24. A method of delivering transgenes to the retinal vasculature of
a mammal comprising intravitreally injecting a transfected lineage
negative hematopoietic stem cell population of claim 14 into the
eye of the mammal.
25. A method of delivering transgenes to the retinal vasculature of
a mammal comprising intravitreally injecting a transfected lineage
negative hematopoietic stem cell population of claim 21 into the
eye of the mammal.
26. A method of rescuing neuronal networks in the eye of a mammal
comprising intravitreally injecting a transfected lineage negative
hematopoietic stem cell population of claim 6 into the eye of the
mammal.
27. A method of rescuing blood vessels in the eye of a mammal
comprising intravitreally injecting a transfected lineage negative
hematopoietic stem cell population of claim 6 into the eye of the
mammal.
28. A method of stimulating upregulation of anti-apoptotic genes in
the eye of a mammal comprising intravitreally injecting a lineage
negative hematopoietic stem cell population of claim 6 into the eye
of the mammal.
29. A method of repairing ischemic tissue in the eye of a mammal
comprising intravitreally injecting a lineage negative
hematopoietic stem cell population of claim 6 into the eye of the
mammal.
30. A method of targeted delivery of stem cells to astrocytes in
the eye of a mammal comprising intravitreally injecting a lineage
negative hematopoietic stem cell population of claim 6 into the eye
of the mammal.
31. A method of targeted delivery of transgenes to astrocytes in
the eye of a mammal comprising intravitreally injecting a lineage
negative hematopoietic stem cell population of claim 14 into the
eye of the mammal.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. patent application
Ser. No. 10/833,743, filed on Apr. 28, 2004, now U.S. Pat. No.
7,838,290, which application is a continuation-in-part of U.S.
patent application Ser. No. 10/628,783, filed on Jul. 25, 2003, now
U.S. Pat. No. 7,153,501, which claims benefit of Provisional Patent
Application No. 60/467,051, filed on May 2, 2003, and Provisional
Patent Application No. 60/398,522, filed on Jul. 25, 2002 the
entire disclosures of which are incorporated herein by
reference
FIELD OF THE INVENTION
[0003] This invention relates to isolated, mammalian, lineage
negative hematopoietic stem cell (Lin.sup.- HSC) populations
derived from bone marrow and their uses. More particularly, the
invention relates to isolated, mammalian, lineage negative
hematopoietic stem cell (Lin.sup.- HSC) populations containing
endothelial progenitor cells (EPC). The invention also relates to
treatment of vascular diseases of the eye by administering
Lin.sup.- HSC and transfected Lin.sup.- HSC populations to the
eye.
BACKGROUND OF THE INVENTION
[0004] Inherited degenerations of the retina affect as many as 1 in
3500 individuals and are characterized by progressive night
blindness, visual field loss, optic nerve atrophy, arteriolar
attenuation, altered vascular permeability and central loss of
vision often progressing to complete blindness (Heckenlively, J.
R., editor, 1988; Retinitis Pigmentosa, Philadelphia: JB Lippincott
Co.). Molecular genetic analysis of these diseases has identified
mutations in over 110 different genes accounting for only a
relatively small percentage of the known affected individuals
(Humphries et al., 1992, Science 256:804-808; Farrar et al. 2002,
EMBO J. 21:857-864.). Many of these mutations are associated with
enzymatic and structural components of the phototransduction
machinery including rhodopsin, cGMP phosphodiesterase, rds
peripherin, and RPE65. Despite these observations, there are still
no effective treatments to slow or reverse the progression of these
retinal degenerative diseases. Recent advances in gene therapy have
led to successful reversal of the rds (Ali et al. 2000, Nat. Genet.
25:306-310) and rd (Takahashi et al. 1999, J. Virol. 73:7812-7816)
phenotypes in mice and the RPE65 phenotype in dogs (Acland et al.
2001, Nat. Genet. 28:92-95) when the wild type transgene is
delivered to photoreceptors or the retinal pigmented epithelium
(RPE) in animals with a specific mutation.
[0005] Age related macular degeneration (ARMD) and diabetic
retinopathy (DR) are the leading causes of visual loss in
industrialized nations and do so as a result of abnormal retinal
neovascularization. Since the retina consists of well-defined
layers of neuronal, glial, and vascular elements, relatively small
disturbances such as those seen in vascular proliferation or edema
can lead to significant loss of visual function. Inherited retinal
degenerations, such as retinitis pigmentosa (RP), are also
associated with vascular abnormalities, such as arteriolar
narrowing and vascular atrophy. While significant progress has been
made in identifying factors that promote and inhibit angiogenesis,
no treatment is currently available to specifically treat ocular
vascular disease.
[0006] For many years it has been known that a population of stem
cells exists in the normal adult circulation and bone marrow.
Different sub-populations of these cells can differentiate along
hematopoietic lineage positive (Lin.sup.+) or lineage negative
(Lin.sup.-) lineages. Furthermore, the lineage negative
hematopoietic stem cell (HSC) population has recently been shown to
contain endothelial progenitor cells (EPC) capable of forming blood
vessels in vitro and in vivo (See Asahara et al. 1997, Science 275:
964-7). These cells can participate in normal and pathological
postnatal angiogenesis (See Lyden et al. 2001 Nat. Med. 7,
1194-201; Kalka et al. 2000, Proc. Natl. Acad. Sci. U.S.A.
97:3422-7; and Kocher et al. 2001, Nat. Med. 7: 430-6) as well as
differentiate into a variety of non-endothelial cell types
including hepatocytes (See Lagasse et al. 2000, Nat. Med.
6:1229-34), microglia (See Priller et al. 2002 Nat. Med.
7:1356-61), cardiomyocytes (See Orlic et al. 2001, Proc. Natl.
Acad. Sci. U.S.A. 98: 10344-9) and epithelium (See Lyden et al.
2001, Nat. Med. 7:1194-1201). Although these cells have been used
in several experimental models of angiogenesis, the mechanism of
EPC targeting to neovasculature is not known and no strategy has
been identified that will effectively increase the number of cells
that contribute to a particular vasculature.
[0007] Hematopoietic stem cells from bone marrow are currently the
only type of stem cell commonly used for therapeutic applications.
Bone marrow HSC's have been used in transplants for over 40 years.
Currently, advanced methods of harvesting purified stem cells are
being investigated to develop therapies for treatment of leukemia,
lymphoma, and inherited blood disorders. Clinical applications of
stem cells in humans have been investigated for the treatment of
diabetes and advanced kidney cancer in limited numbers of human
patients.
SUMMARY OF THE INVENTION
[0008] The present invention provides isolated, mammalian,
population of hematopoietic stem cells (HSCs) that do not express
lineage surface antigens (Lin) on their cell surface, i.e, lineage
negative hematopoietic stem cells (Lin.sup.- HSCs). The Lin.sup.-
HSC populations of the present invention include endothelial
progenitor cells (EPC), also known as endothelial precursor cells,
that selectively target activated retinal astrocytes when
intravitreally injected into the eye. The Lin.sup.- HSCs of the
present invention preferably are derived from adult mammalian bone
marrow, more preferably from adult human bone marrow.
[0009] In a preferred embodiment the Lin.sup.- HSC populations of
the present invention are isolated by extracting bone marrow from
an adult mammal; separating a plurality of monocytes from the bone
marrow; labeling the monocytes with biotin-conjugated lineage panel
antibodies to one or more lineage surface antigens, removing
monocytes that are positive for the lineage surface antigens and
then recovering a Lin.sup.- HSC population containing EPCs.
Preferably the monocytes are labeled with biotin-conjugated lineage
panel antibodies to one or more lineage surface antigen selected
from the group consisting of CD2, CD3, CD4, CD11, CD11a, Mac-1,
CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, Ly-6G, TER-119,
CD45RA, CD56, CD64, CD68, CD86, CD66b, HLA-DR, and CD235a
(Glycophorin A). Preferably, at least about 20% of the cells of the
isolated Lin.sup.- HSC population of the present invention express
the surface antigen CD31.
[0010] The EPC's within the population of Lin.sup.- HSCs of the
present invention extensively incorporate into developing retinal
vessels and remain stably incorporated into neovasculature of the
eye. The isolated, Lin.sup.- HSC populations of the present
invention can be used to rescue and stabilize degenerating retinal
vasculature in mammals, to rescue neuronal networks, and to
facilitate repair of ischemic tissue.
[0011] In one preferred embodiment, the cells of the isolated
Lin.sup.- HSC populations are transfected with a therapeutically
useful gene. For example, the cells can be transfected with
polynucleotides that operably encode for neurotrophic agents or
anti-angiogenic agents that selectively target neovasculature and
inhibit new vessel formation without affecting already established
vessels through a form of cell-based gene therapy. In one
embodiment, the isolated, Lin.sup.- HSC populations of the present
invention include a gene encoding an angiogenesis inhibiting
peptide. The angiogenesis inhibiting Lin.sup.- HSCs are useful for
modulating abnormal blood vessel growth in diseases such as ARMD,
DR and certain retinal degenerations associated with abnormal
vasculature. In another preferred embodiment, the isolated,
Lin.sup.- HSCs of the present invention include a gene encoding a
neurotrophic peptide. The neurotrophic Lin.sup.- HSCs are useful
for promoting neuronal rescue in ocular diseases involving retinal
neural degeneration, such as glaucoma, retinitis pigmentosa, and
the like.
[0012] A particular advantage of ocular treatments with the
isolated Lin.sup.- HSC populations of the present invention is a
vasculotrophic and neurotrophic rescue effect observed in eyes
intravitreally treated with the Lin.sup.- HSCs. Retinal neurons and
photoreceptors are preserved and visual function is maintained in
eyes treated with the isolated Lin.sup.- HSCs of the invention. The
present invention provides a method for treating retinal
degeneration comprising administering isolated Lin.sup.- HSC cells
derived from bone marrow, which contain endothelial progenitor
cells that selectively target activated retinal astrocytes, wherein
at least about 50% the isolated Lin.sup.- HSCs express the surface
antigen CD31 and at least about 50% the isolated Lin.sup.- HSCs
express the surface antigen CD117 (c-kit).
[0013] The present invention also provides a method of isolating
lineage negative hematopoietic stem cell populations containing
endothelial progenitor cells from adult mammalian bone marrow,
preferably from adult human bone marrow. In addition, a line of
genetically identical cells (i.e., clones) can be generated from
human Lin.sup.- HSCs that are useful in regenerative or reparative
treatment of retinal vasculature, as well as for treatment or
amelioration of retinal neuronal tissue degeneration.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 (a and b) depicts schematic diagrams of developing
mouse retina. (a) Development of primary plexus. (b) The second
phase of retinal vessel formation. GCL, ganglion cell layer; IPL,
inner plexus layer; INL, inner nuclear layer; OPL, outer plexus
layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium;
ON, optic nerve; P, periphery.
[0015] FIG. 1c depicts flow cytometric characterization of bone
marrow-derived Lin.sup.+ HSC and Lin.sup.- HSC separated cells. Top
row: Dot plot distribution of non-antibody labeled cells, in which
R1 defines the quantifiable-gated area of positive PE-staining; R2
indicates GFP-positive; Middle row: Lin.sup.- HSC(C57B/6) and
Bottom row: Lin.sup.+ HSC(C57B/6) cells, each cell line labeled
with the PE-conjugated antibodies for Sca-1, c-kit, Flk-1/KDR,
CD31. Tie-2 data was obtained from Tie-2-GFP mice. Percentages
indicate percent of positive-labeled cells out of total Lin.sup.-
HSC or Lin.sup.+ HSC population.
[0016] FIG. 2 depicts engraftment of Lin.sup.- HSCs into developing
mouse retina. (a) At four days post-injection (P6) intravitreally
injected eGFP Lin.sup.- HSC cells attach and differentiate on the
retina (b) Lin.sup.- HSC (B6.129S7-Gtrosa26 mice, stained with
.beta.-gal antibody) establish themselves ahead of the vasculature
stained with collagen IV antibody (asterisk indicates tip of
vasculature). (c) Most of Lin.sup.+ HSC cells (eGFP.sup.+) at four
days post-injection (P6) were unable to differentiate. (d)
Mesenteric eGFP murine EC four days post-injection (P6). (e)
Lin.sup.- HSCs (eGFP.sup.+) injected into adult mouse eyes. (f) Low
magnification of eGFP Lin.sup.- HSCs (arrows) homing to and
differentiating along the pre-existing astrocytic template in the
GFAP-GFP transgenic mouse. (g) Higher magnification of association
between Lin.sup.- cells (eGFP) and underlying astrocyte (arrows).
(h) Non-injected GFAP-GFP transgenic control. (i) Four days
post-injection (P6), eGFP Lin.sup.- HSCs migrate to and undergo
differentiation in the area of the future deep plexus. Left figure
captures Lin.sup.- HSC activity in a whole mounted retina; right
figure indicates location of the Lin.sup.- cells (arrows) in the
retina (top is vitreal side, bottom is scleral side). (j) Double
labeling with .alpha.-CD31-PE and .alpha.-GFP-alexa 488 antibodies.
Seven days after injection, the injected Lin.sup.- HSCs (eGFP),
red) were incorporated into the vasculature (CD31). Arrowheads
indicate the incorporated areas. (k) eGFP Lin.sup.- HSC cells form
vessels fourteen days post-injection (P17). (l and m) Intra-cardiac
injection of rhodamine-dextran indicates that the vessels are
intact and functional in both the primary (l) and deep plexus
(m).
[0017] FIG. 3 (a and b) shows that eGFP Lin.sup.- HSC cells home to
the gliosis (indicated by GFAP expressing-astrocytes, far left
image) induced by both laser (a) and mechanical (b) induced injury
in the adult retina (asterisk indicates injured site). Far right
images are a higher magnification, demonstrating the close
association of the Lin.sup.- HSCs and astrocytes. Calibration
bar=20 .mu.M.
[0018] FIG. 4 shows that Lin.sup.- HSC cells rescue the vasculature
of the retinal degeneration mouse. (a-d) Retinas at 27 days
post-injection (P33) with collagen IV staining; (a) and (b),
retinas injected with Lin.sup.+ HSC cells (Balb/c) showed no
difference in vasculature from normal FVB mice; (c) and (d) retinas
injected with Lin.sup.- HSCs (Balb/c) exhibited a rich vascular
network analogous to a wild-type mouse; (a) and (c), frozen
sections of whole retina (top is vitreal side, bottom is scleral
side) with DAPI staining; (b) and (d), deep plexus of retinal whole
amount; (e) bar graph illustrating the increase in vascularity of
the deep vascular plexus formed in the Lin.sup.- HSC cell-injected
retinas (n=6). The extent of deep retinal vascularization was
quantified by calculating the total length of vessels within each
image. Average total length of vessels/high power field (in
microns) for Lin.sup.- HSC, Lin.sup.+ HSC or control retinas were
compared. (f) Comparison of the length of deep vascular plexus
after injection with Lin.sup.- HSC (R, right eye) or Lin.sup.+ HSC
(L, left eye) cells from rd/rd mouse. The results of six
independent mice are shown (each color represents each mouse). (g)
and (h) Lin.sup.- HSC cells also (Balb/c) rescued the rd/rd
vasculature when injected into P15 eyes. The intermediate and deep
vascular plexus of Lin.sup.- HSC (G) or Lin.sup.+ HSC (H) cell
injected retinas (one month after injection) are shown.
[0019] FIG. 5 depicts photomicrographs of mouse retinal tissue: (a)
deep layer of retinal whole mount (rd/rd mouse), five days
post-injection (P11) with eGFP.sup.+ Lin.sup.- HSCs visible (gray).
(b) and (c) P60 retinal vasculature of Tie-2-GFP (rd/rd) mice that
received Balb/c Lin.sup.- cells (b) or Lin.sup.+ HSC cell (c)
injection at P6. Only endogenous endothelial cells (GFP-stained)
are visible in the left panels of (b) and (c). The middle panels of
(b) and (c) are stained with CD31 antibody; arrows indicate the
vessels stained with CD31 but not with GFP, the right panels of (b)
and (c) show staining with both GFP and CD31. (d) .alpha.-SMA
staining of Lin.sup.- HSC injected (left panel) and control retina
(right panel).
[0020] FIG. 6 shows that T2-TrpRS-transfected Lin.sup.- HSCs
inhibit the development of mouse retinal vasculature. (a) Schematic
representation of human TrpRS, T2-TrpRS and T2-TrpRS with an Igk
signal sequence at the amino terminus. (b) T2-TrpRS transfected
Lin.sup.- HSC-injected retinas express T2-TrpRS protein in vivo.
(1) Recombinant T2-TrpRS produced in E. coli; (2) Recombinant
T2-TrpRS produced in E. coli; (3) Recombinant T2-TrpRS produced in
E. coli; (4) control retina; (5) Lin.sup.- HSC+pSecTag2A (vector
only) injected retina; (6) Lin.sup.- HSC+pKLe135 (Igk-T2-TrpRS in
pSecTag) injected retina. (a) Endogenous TrpRS. (b) Recombinant
T2-TrpRS. (c) T2-TrpRS of Lin.sup.- HSC injected retina. (c-f)
Representative primary (superficial) and secondary (deep) plexuses
of injected retinas, seven days post-injection; (c) and (d) Eyes
injected with empty plasmid-transfected Lin.sup.- HSC developed
normally; (e) and (f) the majority of T2-TrpRS-transfected
Lin.sup.- HSC injected eyes exhibited inhibition of deep plexus;
(c) and (e) primary (superficial) plexus; (d) and (f) secondary
(deep) plexus). Faint outline of vessels observed in (f) are
"bleed-through" images of primary network vessels shown in (e).
[0021] FIG. 7 shows the DNA sequence encoding His.sub.6-tagged
T2-TrpRS, SEQ ID NO: 1.
[0022] FIG. 8 shows the amino acid sequence of His.sub.6-tagged
T2-TrpRS, SEQ ID NO: 2.
[0023] FIG. 9 illustrates photomicrographs and electroretinograms
(ERG) of retinas from mice whose eyes were injected with the
Lin.sup.- HSC of the present invention and with Lin.sup.+ HSC
(controls).
[0024] FIG. 10 depicts statistical plots showing a correlation
between neuronal rescue (y-axis) and vascular rescue x-axis) for
both the intermediate (Int.) and deep vascular layers of rd/rd
mouse eyes treated with Lin.sup.- HSC.
[0025] FIG. 11 depicts statistical plots showing no correlation
between neuronal rescue (y-axis) and vascular rescue x-axis) for
rd/rd mouse eyes that were treated with Lin.sup.+ HSC.
[0026] FIG. 12 is a bar graph of vascular length (y-axis) in
arbitrary relative units for rd/rd mouse eyes treated with the
Lin.sup.- HSC (dark bars) and untreated (light bars) rd/rd mouse
eyes at time points of 1 month (1M), 2 months (2M), and 6 months
(6M) post-injection.
[0027] FIG. 13 includes three bar graphs of the number of nuclei in
the outer neural layer (ONR) of rd/rd mice at 1 month (1M), 2
months (2M) and 6 months (6M), post-injection, and demonstrates a
significant increase in the number of nuclei for eyes treated with
Lin.sup.- HSC (dark bars) relative to control eyes treated with
Lin.sup.+ HSC (light bars).
[0028] FIG. 14 depicts plots of the number of nuclei in the outer
neural layer for individual rd/rd mice, comparing the right eye (R,
treated with Lin.sup.- HSC) relative to the left eye (L, control
eye treated with Lin.sup.+ HSC) at time points (post injection) of
1 month (1M), 2 months (2M), and 6 months (6M); each line in a
given plot compares the eyes of an individual mouse.
[0029] FIG. 15 depicts retinal vasculature and neural cell changes
in rd1/rd1 (C3H/HeJ, left panels) or wild type mice (C57BL/6, right
panels). Retinal vasculature of intermediate (upper panels) or deep
(middle panels) vascular plexuses in whole-mounted retinas (red:
collagen IV, green: CD31) and sections (red: DAPI, green: CD31,
lower panels) of the same retinas are shown (P: postnatal day).
(GCL: ganglion cell layer, INL: inter nuclear layer, ONL: outer
nuclear layer).
[0030] FIG. 16 shows that Lin.sup.- HSC injection rescues the
degeneration of neural cells in rd1/rd1 mice. A, B and C, retinal
vasculature of intermediate (int.) or deep plexus and sections of
Lin.sup.- HSC injected eye (right panels) and contralateral control
cell (CD31.sup.-) injected eye (left panels) at P30 (A), P60 (B),
and P180 (C). D, the average total length of vasculature (+ or -
standard error of the mean) in Lin.sup.- HSC injected or control
cell (CD31.sup.-) injected retinas at P30 (left, n=10), P60
(middle, n=10), and P180 (right, n=6). Data of intermediate (Int.)
and deep vascular plexus are shown separately (Y axis: relative
length of vasculature). E, the average numbers of cell nuclei in
the ONL at P30 (left, n=10), P60 (middle, n=10), or P180 (right,
n=6) of control cell (CD31.sup.-) or Lin.sup.- HSC injected retinas
(Y axis: relative number of cell nuclei in the ONL). F, Linear
correlations between the length of vasculature (X axis) and the
number of cell nuclei in the ONL (Y axis) at P30 (left), P60
(middle), and P180 (right) of Lin.sup.- HSC or control cell
injected retinas.
[0031] FIG. 17 demonstrates that retinal function is rescued by
Lin.sup.- HSC injection. Electroretinographic (ERG) recordings were
used to measure the function of Lin.sup.- HSC or control cell
(CD31.sup.-) injected retinas. A and B, Representative cases of
rescued and non-rescued retinas 2 months after injection. Retinal
section of Lin.sup.- HSC injected right eye (A) and CD31.sup.-
control cell injected left eye (B) of the same animal are shown
(green: CD31 stained vasculature, red: DAPI stained nuclei). C, ERG
results from the same animal shown in A & B.
[0032] FIG. 18 shows that a population of human bone marrow cells
can rescue degenerating retinas in the rd1 mouse (A-C). The rescue
is also observed in another model of retinal degeneration, rd10
(D-K). A, human Lin.sup.- HSCs (hLin.sup.-HSCs) labeled with green
dye can differentiate into retinal vascular cells after
intravitreal injection into C3SnSmn. CB17-Prkdc SCID mice. B and C,
Retinal vasculature (left panels; upper: intermediate plexus,
lower: deep plexus) and neural cells (right panel) in hLin.sup.-
HSC injected eye (B) or contralateral control eye (C) 1.5 months
after injection. D-K, Rescue of rd10 mice by Lin.sup.- HSCs
(injected at P6). Representative retinas at P21 (D: Lin.sup.- HSCs,
H: control cells), P30 (E: Lin.sup.- HSCs, I: control cells), P60
(F: Lin.sup.- HSCs, J: control cells), and P105 (G: Lin.sup.- HSCs,
K: control cells) are shown (treated and control eyes are from the
same animal at each time point). Retinal vasculature (upper image
in each panel is the intermediate plexus; the middle image in each
panel is the deep plexus) was stained with CD31 (green) and
Collagen IV (red). The lower image in each panel shows a cross
section made from the same retina (red: DAPI, green: CD31).
[0033] FIG. 19 demonstrates that crystallin .alpha.A is up
regulated in rescued outer nuclear layer cells after treatment with
Lin.sup.- HSCs but not in contralateral eyes treated with control
cells. Left panel; IgG control in rescued retina, Middle panel;
crystallin .alpha.A in rescued retina, Right panel; crystallin
.alpha.A in non-rescued retina.
[0034] FIG. 20 includes tables of genes that are upregulated in
murine retinas that have been treated with the Lin.sup.- HSCs of
the present invention. (A) Genes whose expression is increased
3-fold in mouse retinas treated with murine Lin.sup.- HSCs. (B)
Crystallin genes that are upregulated in mouse retinas treated with
murine Lin.sup.- HSC. (C) Genes whose expression is increased
2-fold in mouse retinas treated with human Lin.sup.- HSCs. (D)
Genes for neurotrophic factors or growth factors whose expression
is upregulated in mouse retinas treated with human Lin.sup.-
HSCs.
[0035] FIG. 21 illustrates the distribution of CD31 and integrin
.alpha.6 surface antigens on CD133 positive (DC133.sup.+) and CD133
negative (CD133.sup.-) human Lin.sup.- HSC populations of the
present invention.
[0036] FIG. 22 illustrates postnatal retinal development in
wild-type C57/B16 mice raised in normal oxygen levels (normoxia),
at post natal days P0 through P30.
[0037] FIG. 23 illustrates oxygen-induced retinopathy model in
C57/B16 mice raised in high oxygen levels (hyperoxia; 75% oxygen)
between P7 and P12, followed by normoxia from P12-P17.
[0038] FIG. 24 demonstrates vascular rescue by treatment with the
Lin.sup.- HSC populations of the present invention in the
oxygen-induced retinopathy model.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0039] Stem cells are typically identified by the distribution of
antigens on the surface of the cells (for a detailed discussion see
Stem Cells: Scientific Progress and Future Directions, a report
prepared by the National Institutes of Health, Office of Science
Policy, June 2001, Appendix E: Stem Cell Markers, which is
incorporated herein by reference to the extent pertinent).
[0040] Hematopoietic stem cells are that stem cells that are
capable of developing into various blood cell types e.g., B cells,
T cells, granulocytes, platelets, and erythrocytes. The lineage
surface antigens are a group of cell-surface proteins that are
markers of mature blood cell lineages, including CD2, CD3, CD11,
CD11a, Mac-1 (CD11b:CD18), CD14, CD16, CD19, CD24, CD33, CD36,
CD38, CD45, CD45RA, murine Ly-6G, murine TER-119, CD56, CD64, CD68,
CD86 (B7.2), CD66b, human leucocyte antigen DR(HLA-DR), and CD235a
(Glycophorin A). Hematopoietic stem cells that do not express
significant levels of these antigens are commonly referred to a
lineage negative (Lin.sup.-). Human hematopoietic stem cells
commonly express other surface antigens such as CD31, CD34, CD117
(c-kit) and/or CD133. Murine hematopoietic stem cells commonly
express other surface antigens such as CD34, CD117 (c-kit), Thy-1,
and/or Sca-1.
[0041] The present invention provides isolated hematopoietic stem
cells that do not express significant levels of a `lineage surface
antigen` (Lin) on their cell surfaces. Such cells are referred to
herein as `lineage negative` or `Lin.sup.-` hematopoietic stem
cells. In particular this invention provides a population of
Lin.sup.- hematopoietic stems cells (Lin.sup.- HSCs) that include
endothelial progenitor cells (EPCs), which are capable of
incorporating into developing vasculature and then differentiating
to become vascular endothelial cells. Preferably the isolated
Lin.sup.- HSC populations are present in a culture medium such as
phosphate buffered saline (PBS).
[0042] As used herein and in the appended claims, the phrase
`adult` in reference to bone marrow, includes bone marrow isolated
postnatally, i.e., from juvenile and adult individuals, as opposed
to embryos. The term `adult mammal` refers to both juvenile and
fully mature mammals.
[0043] The present invention provides isolated, mammalian, lineage
negative hematopoietic stem cell (Lin.sup.- HSC) populations
containing endothelial progenitor cells (EPCs). The isolated
Lin.sup.- HSC populations of the present invention preferably
comprise mammalian cells in which at least about 20% of the cells
express the surface antigen CD31, which is commonly present on
endothelial cells. In other embodiment, at least about 50% of the
cells express CD31, more preferably at least about 65%, most
preferably at least about 75%. Preferably at least about 50% of the
cells of the Lin.sup.- HSC populations of the present invention
preferably express the integrin .alpha.6 antigen.
[0044] In one preferred murine Lin.sup.- HSC population embodiment,
at least about 50% of the cells express CD31 antigen and at least
about 50% of the cells express the CD117 (c-kit) antigen.
Preferably, at least about 75% of the Lin.sup.- HSC cells express
the surface antigen CD31, more preferably about 81% of the cells.
In another preferred murine embodiment, at least about 65% of the
cells express the surface antigen CD117, more preferably about 70%
of the cells. A particularly preferred embodiment of the present
invention is a population of murine Lin.sup.- HSCs in which about
50% to about 85% of the cells express the surface antigen CD31 and
about 70% to about 75% of the cells express the surface antigen
CD117.
[0045] Another preferred embodiment is a human Lin.sup.- HSC
population in which the cells are CD133 negative, in which at least
about 50% of the cells express the CD31 surface antigen and at
least about 50% of the cells express the integrin .alpha.6 antigen.
Yet another preferred embodiment is a human Lin.sup.- HSC
population in which the cells are CD133 positive, in which at less
than about 30% of the cells express the CD31 surface antigen and
less than about 30% of the cells express the integrin .alpha.6
antigen.
[0046] The isolated Lin.sup.- HSC populations of the present
invention selectively target astrocytes and incorporate into the
retinal neovasculature when intravitreally injected into the eye of
the mammalian species, such as a mouse or a human, from which the
cells were isolated.
[0047] The isolated Lin.sup.- HSC populations of the present
invention include endothelial progenitor cells that differentiate
to endothelial cells and generate vascular structures within the
retina. In particular, the Lin.sup.- HSC populations of the present
invention are useful for the treatment of retinal neovascular and
retinal vascular degenerative diseases, and for repair of retinal
vascular injury. The Lin.sup.- HSC cells of the present invention
promote neuronal rescue in the retina and promote upregulation of
anti-apoptotic genes. It has surprisingly been found that adult
human Lin.sup.- HSC cells of the present invention can inhibit
retinal degeneration even in severe combined immunodeficient (SCID)
mice suffering from retinal degeneration. Additionally, the
Lin.sup.- HSC populations can be utilized to treat retinal defects
in the eyes of neonatal mammals, such as mammals suffering from
oxygen induced retinopathy or retinopathy of prematurity.
[0048] The present invention also provides a method of treating
ocular diseases in a mammal comprising isolating from the bone
marrow of the mammal a lineage negative hematopoietic stem cell
population that includes endothelial progenitor cells, and
intravitreally injecting the isolated stem cells into an eye of the
mammal in a number sufficient to arrest the disease. The present
method can be utilized to treat ocular diseases such as retinal
degenerative diseases, retinal vascular degenerative diseases,
ischemic retinopathies, vascular hemorrhages, vascular leakage, and
choroidopathies in neonatal, juvenile or fully mature mammals.
Examples of such diseases include age related macular degeneration
(ARMD), diabetic retinopathy (DR), presumed ocular histoplasmosis
(POHS), retinopathy of prematurity (ROP), sickle cell anemia, and
retinitis pigmentosa, as well as retinal injuries.
[0049] The number of stem cells injected into the eye is sufficient
for arresting the disease state of the eye. For example, the number
of cells can be effective for repairing retinal damage of the eye,
stabilizing retinal neovasculature, maturing retinal
neovasculature, and preventing or repairing vascular leakage and
vascular hemorrhage.
[0050] Cells of the Lin.sup.- HSC populations of the present
invention can be transfected with therapeutically useful genes,
such as genes encoding antiangiogenic proteins for use in ocular,
cell-based gene therapy and genes encoding neurotrophic agents to
enhance neuronal rescue effects.
[0051] The transfected cells can include any gene which is
therapeutically useful for treatment of retinal disorders. In one
preferred embodiment, the transfected Lin.sup.- HSCs of the present
invention include a gene operably encoding an antiangiogenic
peptide, including proteins, or protein fragments such as TrpRS or
antiangiogenic fragments thereof, e.g., the T1 and T2 fragments of
TrpRS, which are described in detail in co-owned, co-pending U.S.
patent application Ser. No. 10/080,839, the disclosure of which is
incorporated herein by reference. The transfected Lin.sup.- HSCs
encoding an antiangiogenic peptide of the present invention are
useful for treatment of retinal diseases involving abnormal
vascular development, such as diabetic retinopathy, and like
diseases. Preferably the Lin.sup.- HSCs are human cells.
[0052] In another preferred embodiment, the transfected Lin.sup.-
HSCs of the present invention include a gene operably encoding a
neurotrophic agent such as nerve growth factor, neurotrophin-3,
neurotrophin-4, neurotrophin-5, ciliary neurotrophic factor,
retinal pigmented epithelium-derived neurotrophic factor,
insulin-like growth factor, glial cell line-derived neurotrophic
factor, brain-derived neurotrophic factor, and the like. Such
neurotrophic Lin.sup.- HSCs are useful for promoting neuronal
rescue in retinal neuronal degenerative diseases such as glaucoma
and retinitis pigmentosa, in treatment of injuries to the retinal
nerves, and the like. Implants of ciliary neurotrophic factor have
been reported as useful for the treatment of retinitis pigmentosa
(see Kirby et al. 2001, Mol. Ther. 3(2):241-8; Farrar et al. 2002,
EMBO Journal 21:857-864). Brain-derived neurotrophic factor
reportedly modulates growth associated genes in injured retinal
ganglia (see Fournier, et al., 1997, J. Neurosci. Res. 47:561-572).
Glial cell line derived neurotrophic factor reportedly delays
photoreceptor degeneration in retinitis pigmentosa (see McGee et
al. 2001, Mol. Ther. 4(6):622-9).
[0053] The present invention also provides a method of isolating a
lineage negative hematopoietic stem cells comprising endothelial
progenitor cells from bone marrow of a mammal. The method entails
the steps of (a) extracting bone marrow from an adult mammal; (b)
separating a plurality of monocytes from the bone marrow; (c)
labeling the monocytes with biotin-conjugated lineage panel
antibodies to one or more lineage surface antigens, preferably
lineage surface antigens selected from the group consisting of CD2,
CD3, CD4, CD11, CD11a, Mac-1, CD14, CD16, CD19, CD24, CD33, CD36,
CD38, CD45, Ly-6G (murine), TER-119 (murine), CD45RA, CD56, CD64,
CD68, CD86 (B7.2), CD66b, human leucocyte antigen DR(HLA-DR), and
CD235a (Glycophorin A); (d) removing monocytes that are positive
for said one or more lineage surface antigens from the plurality of
monocytes and recovering a population of lineage negative
hematopoietic stem cells containing endothelial progenitor cells,
preferably in which at least about 20% of the cells express
CD31.
[0054] When the Lin.sup.- HSC are isolated from adult human bone
marrow, preferably the monocytes are labeled with biotin-conjugated
lineage panel antibodies to lineage surface antigens CD2, CD3, CD4,
CD11a, Mac-1, CD14, CD16, CD19, CD33, CD38, CD45RA, CD64, CD68,
CD86 (B7.2), and CD235a. When the Lin.sup.- HSC are isolated from
adult murine bone marrow, preferably the monocytes are labeled with
biotin-conjugated lineage panel antibodies to lineage surface
antigens CD3, CD11, CD45, Ly-6G, and TER-119.
[0055] In a preferred method, the cells are isolated from adult
human bone marrow and are further separated by CD133 lineage. One
preferred method of isolating human Lin.sup.- HSCs includes the
additional steps of labeling the monocytes with a biotin-conjugated
CD133 antibody and recovering a CD133 positive, Lin.sup.- HSC
population. Typically, less than about 30% of such cells express
CD31 and less than about 30% of such cell express integrin
.alpha.6. The human Cd133 positive, Lin.sup.- HSC populations of
the present invention can target sites of peripheral
ischemia-driven neovascularization when injected into eyes that are
not undergoing angiogenesis.
[0056] Another preferred method of isolating human Lin.sup.- HSCs
includes the additional steps of labeling the monocytes with a
biotin-conjugated CD133 antibody, removing CD133 positive cells,
and recovering a CD133 negative, Lin.sup.- HSC population.
Typically, at least about 50% of such cells express CD31 and at
least about 50% of such cell express integrin .alpha.6. The human
CD133 negative, Lin.sup.- HSC populations of the present invention
can incorporate into developing vasculature when injected into eyes
that are undergoing angiogenesis.
[0057] The present invention also provides methods for treating
ocular angiogenic diseases by administering transfected Lin.sup.-
HSC cells of the present invention by intravitreal injection of the
cells into the eye. Such transfected Lin.sup.- HSC cells comprise
Lin.sup.- HSC transfected with a therapeutically useful gene, such
as a gene encoding antiangiogenic or neurotrophic gene product.
Preferably the transfected Lin.sup.- HSC cells are human cells.
[0058] Preferably, at least about 1.times.10.sup.5 Lin.sup.- HSC
cells or transfected Lin.sup.- HSC cells are administered by
intravitreal injection to a mammalian eye suffering from a retinal
degenerative disease. The number of cells to be injected may depend
upon the severity of the retinal degeneration, the age of the
mammal and other factors that will be readily apparent to one of
ordinary skill in the art of treating retinal diseases. The
Lin.sup.- HSC may be administered in a single dose or by multiple
dose administration over a period of time, as determined by the
clinician in charge of the treatment.
[0059] The Lin.sup.- HSCs of the present invention are useful for
the treatment of retinal injuries and retinal defects involving an
interruption in or degradation of the retinal vasculature or
retinal neuronal degeneration. Human Lin.sup.- HSCs also can be
used to generate a line of genetically identical cells, i.e.,
clones, for use in regenerative or reparative treatment of retinal
vasculature, as well as for treatment or amelioration of retinal
neuronal degeneration.
METHODS
Example 1
Cell Isolation and Enrichment; Preparation of Murine Lin.sup.- HSC
Populations A and B
[0060] General Procedure. All in vivo evaluations were performed in
accordance with the NIH Guide for the Care and Use of Laboratory
Animals, and all evaluation procedures were approved by The Scripps
Research Institute (TSR1, La Jolla, Calif.) Animal Care and Use
Committee. Bone marrow cells were extracted from B6.129S7-Gtrosa26,
Tie-2GFP, ACTbEGFP, FVB/NJ (rd/rd mice) or Balb/cBYJ adult mice
(The Jackson Laboratory, ME).
[0061] Monocytes were then separated by density gradient separation
using HISTOPAQUE.TM. polysucrose gradient (Sigma, St. Louis, Mo.)
and labeled with biotin conjugated lineage panel antibodies (CD45,
CD3, Ly-6G, CD11, TER-119, Pharmingen, San Diego, Calif.) for
Lin.sup.- selection in mice. Lineage positive (Lin.sup.+) cells
were separated and removed from Lin.sup.- HSC using a magnetic
separation device (AUTOMACS.TM. sorter, Miltenyi Biotech, Auburn,
Calif.). The resulting Lin.sup.- HSC population, containing
endothelial progenitor cells was further characterized using a
FACS.TM. Calibur flow cytometer (Becton Dickinson, Franklin Lakes,
N.J.) using following antibodies: PE-conjugated-Sca-1, c-kit, KDR,
and CD31 (Pharmingen, San Diego, Calif.). Tie-2-GFP bone marrow
cells were used for characterization of Tie-2.
[0062] To harvest adult mouse endothelial cells, mesenteric tissue
was surgically removed from ACTbEGFP mouse and placed in
collagenase (Worthington, Lakewood, N.J.) to digest the tissue,
followed by filtration using a 45 .mu.m filter. Flow-through was
collected and incubated with Endothelial Growth Media (Clonetics,
San Diego, Calif.). Endothelial characteristics were confirmed by
observing morphological cobblestone appearance, staining with CD31
mAb (Pharmingen) and examining cultures for the formation of
tube-like structures in MATRIGEL.TM. matrix (Beckton Dickinson,
Franklin Lakes, N.J.).
[0063] Murine Lin.sup.- HSC Population A. Bone marrow cells were
extracted from ACTbEGFP mice by the General Procedure described
above. The Lin.sup.- HSC cells were characterized by FACS flow
cytometry for CD31, c-kit, Sca-1, Flk-1, and Tie-2 cell surface
antigen markers. The results are shown in FIG. 1c. About 81% of the
Lin.sup.- HSC exhibited the CD31 marker, about 70.5% of the
Lin.sup.- HSC exhibited the c-kit marker, about 4% of the Lin.sup.-
HSC exhibited the Sca-1 marker, about 2.2% of the Lin.sup.- HSC
exhibited the Flk-1 marker and about 0.91% of the Lin.sup.- HSC
cell exhibited the Tie-2 marker. In contrast, the Lin.sup.+ HSC
that were isolated from these bone marrow cells had a significantly
different cell marker profile (i.e., CD31: 37.4%; c-kit: 20%;
Sca-1: 2.8%; Flk-: 0.05%).
[0064] Murine Lin.sup.-HSC Population B. Bone marrow cells were
extracted from Balb/C, ACTbEGFP, and C3H mice by the General
Procedure described above. The Lin.sup.- HSC cells were analyzed
for the presence of cell surface markers (Sca-1, KDR, c-kit, CD34,
CD31 and various integrins: .alpha.1, .alpha.2, .alpha.3, .alpha.4,
.alpha.5, .alpha.6, .alpha..sub.M, .alpha..sub.V, .alpha..sub.X,
.alpha..sub.IIb, .beta..sub.1, .beta..sub.4, .beta..sub.3,
.beta..sub.4, .beta..sub.5 and .beta..sub.7). The results are shown
in Table 1.
TABLE-US-00001 TABLE 1 Characterization of Lin.sup.- HSC Population
B. Cell Marker Lin.sup.- HSC .alpha.1 0.10 .alpha.2 17.57 .alpha.3
0.22 .alpha.4 89.39 .alpha.5 82.47 .alpha.6 77.70 .alpha.L 62.69
.alpha.M 35.84 .alpha.X 3.98 .alpha.V 33.64 .alpha.IIb 0.25 .beta.1
86.26 .beta.2 49.07 .beta.3 45.70 .beta.4 0.68 .beta.5 9.44 .beta.7
11.25 CD31 51.76 CD34 55.83 Flk-1/KDR 2.95 c-kit (CD117) 74.42
Sca-1 7.54
Example 2
Intravitreal Administration of Cells in a Murine Model
[0065] An eyelid fissure was created in a mouse eyelid with a fine
blade to expose the P2 to P6 eyeball. Lineage negative HSC
Population A of the present invention (approximately 10.sup.5 cells
in about 0.5 .mu.l to about 1 .mu.l of cell culture medium) was
then injected intravitreally using a 33-gauge (Hamilton, Reno,
Nev.) needled-syringe.
Example 3
EPC Transfection
[0066] Murine Lin.sup.- HSC (Population A) were transfected with
DNA encoding the T2 fragment of TrpRS also enclosing a His.sub.6
tag (SEQ ID NO: 1, FIG. 7) using FuGENE.TM.6 Transfection Reagent
(Roche, Indianapolis, Ind.) according to manufacturer's protocol.
Lin.sup.- HSC cells (about 10.sup.6 cell per ml) were suspended in
opti-MEM.TM. medium (Invitrogen, Carlsbad, Calif.) containing stem
cell factor (PeproTech, Rocky Hill, N.J.). DNA (about 1 .mu.g) and
FuGENE reagent (about 3 .mu.l) mixture was then added, and the
mixtures were incubated at about 37.degree. C. for about 18 hours.
After incubation, cells were washed and collected. The transfection
rate of this system was approximately 17% that was confirmed by
FACS analysis. T2 production was confirmed by western blotting. The
amino acid sequence of His.sub.6-tagged T2-TrpRS is shown as SEQ ID
NO: 2, FIG. 8.
Example 4
Immunohistochemistry and Confocal Analysis
[0067] Mouse retinas were harvested at various time points and were
prepared for either whole mounting or frozen sectioning. For whole
mounts, retinas were fixed with 4% paraformaldehyde, and blocked in
50% fetal bovine serum (FBS) and 20% normal goat serum for one hour
at ambient room temperature. Retinas were processed for primary
antibodies and detected with secondary antibodies. The primaries
used were: anti-Collagen IV (Chemicon, Temecula, Calif.,
anti-.beta.-gal (Promega, Madison, Wis.), anti-GFAP (Dako
Cytomation, Carpenteria, Calif.), anti-.alpha.-smooth muscle actin
(.alpha.-SMA, Dako Cytomation). Secondary antibodies used were
conjugated either to Alexa 488 or 594 fluorescent markers
(Molecular Probes, Eugene, Oreg.). Images were taken using an MRC
1024 Confocal microscope (Bio-Rad, Hercules, Calif.).
Three-dimensional images were created using LASERSHARP.TM. software
(Bio-Rad) to examine the three different layers of vascular
development in the whole mount retina. The difference in GFP pixel
intensity between enhanced GFP (eGFP) mice and GFAP/wtGFP mice,
distinguished by confocal microscopy, was utilized to create the 3D
images.
Example 5
In vivo Retinal Angiogenesis Quantification Assay in Mice
[0068] For T2-TrpRS analysis, the primary and deep plexus were
reconstructed from the three dimensional images of mouse retinas.
The primary plexus was divided into two categories: normal
development, or halted vascular progression. The categories of
inhibition of deep vascular development were construed based upon
the percentage of vascular inhibition including the following
criteria: complete inhibition of deep plexus formation was labeled
`Complete`, normal vascular development (including less than 25%
inhibition) was labeled `Normal` and the remainder labeled
`Partial.` For the rd/rd mouse rescue data, four separate areas of
the deeper plexus in each whole mounted retina were captured using
a 10.times. lens. The total length of vasculature was calculated
for each image, summarized and compared between the groups. To
acquire accurate information, Lin.sup.- HSC were injected into one
eye and Lin.sup.+ HSC into another eye of the same mouse.
Non-injected control retinas were taken from the same litter.
Example 6
Adult Retinal Injury Murine Models
[0069] Laser and scar models were created using either a diode
laser (150 mW, 1 second, 50 mm) or mechanically by puncturing the
mouse retina with a 27 gauge needle. Five days after injury, cells
were injected using the intravitreal method. Eyes were harvested
from the mice five days later.
Example 7
Neurotrophic Rescue of Retinal Regeneration
[0070] Adult murine bone marrow derived lineage negative
hematopoietic stem cells (Lin.sup.- HSC) have a vasculotrophic and
neurotrophic rescue effect in a mouse model of retinal
degeneration. Right eyes of 10-day old mice were injected
intravitreally with about 0.5 microliters containing about 10.sup.5
Lin.sup.- HSC of the present invention and evaluated 2 months later
for the presence of retinal vasculature and neuronal layer nuclear
count. The left eyes of the same mice were injected with about the
same number of Lin.sup.+ HSC as a control, and were similarly
evaluated. As shown in FIG. 9, in the Lin.sup.- HSC treated eyes,
the retinal vasculature appeared nearly normal, the inner nuclear
layer was nearly normal and the outer nuclear layer (ONL) had about
3 to about 4 layers of nuclei. In contrast, the contralateral
Lin.sup.+ HSC treated eye had a markedly atrophic middle retinal
vascular layer, a completely atrophic outer retinal vascular layer;
the inner nuclear layer was markedly atrophic and the outer nuclear
layer was completely gone. This was dramatically illustrated in
Mouse 3 and Mouse 5. In Mouse 1, there was no rescue effect and
this was true for approximately 15% of the injected mice.
[0071] When visual function was assessed with electroretinograms
(ERG), the restoration of a positive ERG was observed when both the
vascular and neuronal rescue was observed (Mice 3 and 5). Positive
ERG was not observed when there was no vascular or neuronal rescue
(Mouse 1). This correlation between vascular and neurotrophic
rescue of the rd/rd mouse eyes by the Lin.sup.- HSC of the present
invention is illustrated by a regression analysis plot shown in
FIG. 10. A correlation between neuronal (y-axis) and vascular
.alpha.-axis) recovery was observed for the intermediate
vasculature type (r=0.45) and for the deep vasculature
(r=0.67).
[0072] FIG. 11 shows the absence of any statistically significant
correlation between vascular and neuronal rescue by Lin.sup.+ HSC.
The vascular rescue was quantified and the data are presented in
FIG. 12. Data for mice at 1 month (1M), 2 months (2M), and 6 months
(6M), post-injection shown in FIG. 12, demonstrate that vascular
length was significantly increased in eyes treated with the
Lin.sup.- HSC of the present invention (dark bars) relative to the
vascular length in untreated eyes from the same mouse (light bars),
particularly at 1 month and 2 months, post-injection. The
neurotrophic rescue effect was quantified by counting nuclei in the
inner and outer nuclear layers about two months after injection of
Lin.sup.- HSC or Lin.sup.+ HSC. The results are presented in FIGS.
13 and 14.
Example 8
Human Lin.sup.- HSC Population
[0073] Bone marrow cells were extracted from healthy adult human
volunteers by the General Procedure described above. Monocytes were
then separated by density gradient separation using HISTOPAQUE.TM.
polysucrose gradient (Sigma, St. Louis, Mo.). To isolate the
Lin.sup.- HSC population from human bone marrow mononuclear cells
the following biotin conjugated lineage panel antibodies were used
with the magnetic separation system (AUTOMACS.TM. sorter, Miltenyi
Biotech, Auburn, Calif.): CD2, CD3, CD4, CD11a, Mac-1, CD14, CD16,
CD19, CD33, CD38, CD45RA, CD64, CD68, CD86, CD235a
(Pharmingen).
[0074] The human Lin.sup.- HSC population was further separated
into two sub-populations based on CD133 expression. The cells were
labeled with biotin-conjugated CD133 antibodies ans separated into
CD133 positive and CD133 negative sub-populations.
Example 9
Intravitreal Administration of Human and Murine Cells in Murine
Models for Retinal Degeneration
[0075] C3H/HeJ, C3SnSmn.CB17-Prkdc SCID, and rd10 mouse strains
were used as retinal degeneration models. C3H/HeJ and
C3SnSmn.CB17-Prkdc SCID mice (The Jackson Laboratory, Maine) were
homozygous for the retinal degeneration 1 (rd1) mutation, a
mutation that causes early onset severe retinal degeneration. The
mutation is located in exon 7 of the Pde6b gene encoding the rod
photoreceptor cGMP phosphodiesterase .beta. subunit. The mutation
in this gene has been found in human patients with autosomal
recessive retinitis pigmentosa (RP). C3SnSmn.CB17-Prkdc SCID mice
are also homozygous for the severe combined immune deficiency
spontaneous mutation (Prkdc SCID) and were used for human cell
transfer experiments. Retinal degeneration in rd10 mice is caused
by a mutation in exon 13 of Pde6b gene. This is also a clinically
relevant RP model with later onset and milder retinal degeneration
than rd1/rd1). All evaluations were performed in accordance with
the NIH Guide for the Care and Use of Laboratory Animals, and all
procedures were approved by The Scripps Research Institute Animal
Care and Use Committee.
[0076] An eyelid fissure was created in a mouse eyelid with a fine
blade to expose the P2 to P6 eyeball. Lineage negative HSC cells
for murine population A or human population C (approximately
10.sup.5 cells in about 0.5 .mu.l to about 1 .mu.l of cell culture
medium) were then injected in the mouse eye intravitreally using a
33-gauge (Hamilton, Reno, Nev.) needled-syringe. To visualize the
injected human cells, cells were labeled with dye (Cell tracker
green CMFDA, Molecular Probes) before injection.
[0077] Retinas were harvested at various time points and fixed with
4% paraformaldehyde (PFA) and methanol followed by blocking in 50%
FBS/20% NGS for one hour at room temperature. To stain retinal
vasculature, retinas were incubated with anti-CD31 (Pharmingen) and
anti-collagen IV (Chemicon) antibodies followed by Alexa 488 or 594
conjugated secondary antibodies (Molecular Probes, Eugene, Oreg.).
The retinas were laid flat with four radial relaxing incisions to
obtain a whole mount preparation. Images of vasculature in
intermediate or deep retinal vascular plexuses (see Dorrell et al.
2002 Invest Ophthalmol. Vis. Sci. 43:3500-3510) were obtained using
a Radiance MP2100 confocal microscope and LASERSHARP.TM. software
(Biorad, Hercules, Calif.). For quantification of vasculature, four
independent fields (900 .mu.m.times.900 .mu.m) were chosen randomly
from the mid portion of the intermediate or deep vascular layer and
the total length of vasculature was measured using LASERPIX.TM.
analyzing software (Biorad). The total lengths of these four fields
in the same plexus were used for further analysis.
[0078] The flat-mounted retinas were re-embedded for cryostat
sections. Retinas were placed in 4% PFA overnight followed by
incubation with 20% sucrose. The retinas were embedded in optimal
cutting temperature compound (OCT: Tissue-Tek; Sakura FineTech,
Torrance, Calif.). Cryostat sections (10 .mu.m) were re-hydrated in
PBS containing the nuclear dye DAPI (Sigma-Aldrich, St. Louis,
Mo.). DAPI-labeled nuclear images of three different areas (280
.mu.m width, unbiased sampling) in a single section that contained
optic nerve head and the entire peripheral retina were taken by
confocal microscope. The numbers of the nuclei located in ONL of
the three independent fields in one section were counted and summed
up for analysis. Simple linear-regression analysis was performed to
examine the relationship between the length of vasculature in the
deep plexus and the number of cell nuclei in the ONL.
[0079] Following overnight dark-adaptation, mice were anesthetized
by intraperitoneal injection of 15 .mu.g/gm ketamine and 7 .mu.g/gm
xylazine. Electroretinograms (ERGs) were recorded from the corneal
surface of each eye after pupil dilation (1% atropine sulfate)
using a gold loop corneal electrode together with a mouth reference
and tail ground electrode. Stimuli were produced with a Grass
Photic Stimulator (PS33 Plus, Grass Instruments, Quincy, Mass.)
affixed to the outside of a highly reflective Ganzfeld dome. Rod
responses were recorded to short-wavelength (Wratten 47A;
.lamda..sub.max=470 nm) flashes of light over a range of
intensities up to the maximum allowable by the photic stimulator
(0.668 cd-s/m.sup.2). Response signals were amplified (CP511 AC
amplifier, Grass Instruments), digitized (PCI-1200, National
Instruments, Austin, Tex.) and computer-analyzed. Each mouse served
as its own internal control with ERGs recorded from both the
treated and untreated eyes. Up to 100 sweeps were averaged for the
weakest signals. The averaged responses from the untreated eye were
digitally subtracted from the responses from the treated eye and
this difference in signal was used to index functional rescue.
[0080] Microarray analysis was used for evaluation of Lin.sup.-
HSC-targeted retinal gene expression. P6 rd/rd mice were injected
with either Lin.sup.- or CD31.sup.- HSCs. The retinas of these mice
were dissected 40 days post-injection in RNase free medium (rescue
of the retinal vasculature and the photoreceptor layer is obvious
at this time point after injection). One quadrant from each retina
was analyzed by whole mount to ensure that normal HSC targeting as
well as vasculature and neural protection had been achieved. RNA
from retinas with successful injections was purified using a TRIzol
(Life Technologies, Rockville, Md.), phenol/chloroform RNA
isolation protocol. RNA was hybridized to Affymetrix Mu74Av2 chips
and gene expression was analyzed using GENESPRING.TM. software
(SiliconGenetics, Redwood City, Calif.). Purified human or mouse
HSCs were injected intravitreally into P6 mice. At P45 the retinas
were dissected and pooled into fractions of 1) human HSC-injected,
rescued mouse retinas, 2) human HSC-injected, non-rescued mouse
retinas, and 3) mouse HSC-injected, rescued mouse retinas for
purification of RNA and hybridization to human-specific U133A
Affymetrix chips. GENESPRING.TM. software was used to identify
genes that were expressed above background and with higher
expression in the human HSC-rescued retinas. The probe-pair
expression profiles for each of these genes were then individually
analyzed and compared to a model of normal human U133A microarray
experiments using dChip to determine human species specific
hybridization and to eliminate false positives due to cross-species
hybridization.
[0081] When the CD133 positive and CD133 negative Lin.sup.- HSC
sub-population was intravitreally injected into the eyes of
neonatal SCID mice, the greatest extent of incorporation into the
developing vasculature was observed for the CD133 negative
sub-population, which expresses both CD31 and integrin .alpha.6
surface antigens (see FIG. 21, bottom). The CD133 positive
sub-population, which does not express CD31 or integrin .alpha.6
(FIG. 21, top) appears to target sites of peripheral
ischemia-driven neovascularization, but not when injected into eyes
undergoing angiogenesis.
Example 10
Intravitreal Administration of Murine Cells in Murine Models for
Oxygen Induced Retinal Degeneration
[0082] New born wild-type C57B16 mice were exposed to hyperoxia
(75% oxygen) between postnatal days P7 to P12 in an oxygen-induced
retinal degeneration (OIR) model. FIG. 22 illustrates normal
postnatal vascular development in C57B16 mice from P0 to P30. At P0
only budding superficial vessels can be observed around the optic
disc. Over the next few days, the primary superficial network
extends toward the periphery, reaching the far periphery by day
P10. Between P7 and P12, the secondary (deep) plexus develops. By
P17, an extensive superficial and deep network of vessels is
present (FIG. 22, insets). In the ensuing days, remodeling occurs
along with development of the tertiary (intermediate) layer of
vessels until the adult structure is reached approximately at
P21.
[0083] In contrast, in the OIR model, following exposure to 75%
oxygen at P7-P12, the normal sequence of events is severely
disrupted (FIG. 23). Adult murine Lin.sup.- HSC populations of the
invention were intravitreally injected at P3 in an eye of a mouse
that was subsequently subjected to OIR, the other eye was injected
with PBS or CD31 negative cells as a control. FIG. 24 illustrates
that the Lin.sup.- HSC populations of the present invention can
reverse the degenerative effects of high oxygen levels in the
developing mouse retina. Fully developed superficial and deep
retinal vasculature was observed at P17 in the treated eyes,
whereas in the control eyes showed large avascular areas with
virtually no deep vessels (FIG. 24). Approximately 100 eyes of mice
in the OIR model were observed. Normal vascularization was observed
in 58% of the eyes treated with the Lin.sup.- HSC populations of
the invention, compared to 12% of the control eyes treated with
CD31.sup.- cells and 3% of the control eyes treated with PBS.
RESULTS
Murine Retinal Vascular Development; A Model for Ocular
Angiogenesis
[0084] The mouse eye provides a recognized model for the study of
mammalian retinal vascular development, such as human retinal
vascular development. During development of the murine retinal
vasculature, ischemia-driven retinal blood vessels develop in close
association with astrocytes. These glial elements migrate onto the
third trimester human fetus, or the neonatal rodent, retina from
the optic disc along the ganglion cell layer and spread radially.
As the murine retinal vasculature develops, endothelial cells
utilize this already established astrocytic template to determine
the retinal vascular pattern (See FIGS. 1a and b). FIG. 1 (a and b)
depicts schematic diagrams of developing mouse retina. FIG. 1a
depicts development of the primary plexus (dark lines at upper left
of the diagram) superimposed over the astrocyte template (light
lines) whereas, FIG. 1b depicts the second phase of retinal vessel
formation. In the Figures, GCL stands for ganglion cell layer; IPL
stands for inner plexus layer; INL stands for inner nuclear layer;
OPL stands for outer plexus layer; ONL stands for outer nuclear
layer; RPE stands for retinal pigment epithelium; ON stands for
optic nerve; and P stands for periphery.
[0085] At birth, retinal vasculature is virtually absent. By
postnatal day 14 (P14) the retina has developed complex primary
(superficial) and secondary (deep) layers of retinal vessels
coincident with the onset of vision. Initially, spoke-like
peripapillary vessels grow radially over the pre-existing
astrocytic network towards the periphery, becoming progressively
interconnected by capillary plexus formation. These vessels grow as
a monolayer within the nerve fiber through P10 (FIG. 1a). Between
P7-P8 collateral branches begin to sprout from this primary plexus
and penetrate into the retina to the outer plexiform layer where
they form the secondary, or deep, retinal plexus. By P21, the
entire network undergoes extensive remodeling and a tertiary, or
intermediate, plexus forms at the inner surface of inner nuclear
layer (FIG. 1b).
[0086] The neonatal mouse retinal angiogenesis model is useful for
studying the role of HSC during ocular angiogenesis for several
reasons. In this physiologically relevant model, a large astrocytic
template exists prior to the appearance of endogenous blood
vessels, permitting an evaluation of the role for cell-cell
targeting during a neovascular process. In addition, this
consistent and reproducible neonatal retinal vascular process is
known to be hypoxia-driven, in this respect having similarities to
many retinal diseases in which ischemia is known to play a
role.
Enrichment of Endothelial Progenitor Cells (EPC) From Bone
Marrow
[0087] Although cell surface marker expression has been extensively
evaluated on the EPC population found in preparations of HSC,
markers that uniquely identify EPC are still poorly defined. To
enrich for EPC, hematopoietic lineage marker positive cells
(Lin.sup.+), i.e., B lymphocytes (CD45), T lymphocytes (CD3),
granulocytes (Ly-6G), monocytes (CD11), and erythrocytes (TER-119),
were depleted from bone marrow mononuclear cells of mice. Sca-1
antigen was used to further enrich for EPC. A comparison of results
obtained after intravitreal injection of identical numbers of
either Lin.sup.- Sca-1.sup.+ cells or Lin.sup.- cells, no
difference was detected between the two groups. In fact, when only
Lin.sup.- Sca-1.sup.- cells were injected, far greater
incorporation into developing blood vessels was observed.
[0088] The Lin.sup.- HSC populations of the present invention are
enriched with EPCs, based on functional assays. Furthermore,
Lin.sup.+ HSC populations functionally behave quite differently
from the Lin.sup.- HSC populations. Epitopes commonly used to
identify EPC for each fraction (based on previously reported in
vitro characterization studies) were also evaluated. While none of
these markers were exclusively associated with the Lin.sup.-
fraction, all were increased about 70 to about 1800% in the
Lin.sup.- HSC, compared to the Lin.sup.+ HSC fraction (FIG. 1c).
FIG. 1c illustrates flow cytometric characterization of bone
marrow-derived Lin.sup.+ HSC and Lin.sup.- HSC separated cells. The
top row of FIG. 1c shows a hematopoietic stem cell dot plot
distribution of non-antibody labeled cells. R1 defines the
quantifiable-gated area of positive PE-staining; R2 indicates
GFP-positive. Dot plots of Lin.sup.- HSC are shown in the middle
row and dot plots of Lin.sup.+ HSC are shown in the bottom row. The
C57B/6 cells were labeled with the PE-conjugated antibodies for
Sca-1, c-kit, Flk-1/KDR, CD31. Tie-2 data was obtained from
Tie-2-GFP mice. The percentages in the corners of the dot plots
indicate the percent of positive-labeled cells out of total
Lin.sup.- or Lin.sup.+ HSC population. Interestingly, accepted EPC
markers like Flk-1/KDR, Tie-2, and Sca-1 were poorly expressed and,
thus, not used for further fractionation.
Intravitreally Injected HSC Lin.sup.- Cells Contain EPC That Target
Astrocytes and Incorporate into Developing Retinal Vasculature
[0089] To determine whether intravitreally injected Lin.sup.- HSC
can target specific cell types of the retina, utilize the
astrocytic template and participate in retinal angiogenesis,
approximately 10.sup.5 cells from a Lin.sup.- HSC composition of
the present invention or Lin.sup.+ HSC cells (control, about
10.sup.5 cells) isolated from the bone marrow of adult (GFP or LacZ
transgenic) mice were injected into postnatal day 2 (P2) mouse
eyes. Four days after injection (P6), many cells from the Lin.sup.-
HSC composition of the present invention, derived from GFP or LacZ
transgenic mice were adherent to the retina and had the
characteristic elongated appearance of endothelial cells (FIG. 2a).
FIG. 2 illustrates engraftment of Lin cells into developing mouse
retina. As shown in FIG. 2a, the four days post-injection (P6)
intravitreally injected eGFP+Lin.sup.- HSC attach and differentiate
on the retina.
[0090] In many areas of the retinas, the GFP-expressing cells were
arranged in a pattern conforming to underlying astrocytes and
resembled blood vessels. These fluorescent cells were observed
ahead of the endogenous, developing vascular network (FIG. 2b).
Conversely, only a small number of Lin.sup.+ HSC (FIG. 2c), or
adult mouse mesenteric endothelial cells (FIG. 2d) attached to the
retinal surface. In order to determine whether cells from an
injected Lin.sup.- HSC population could also attach to retinas with
already established vessels, we injected a Lin.sup.- HSC
composition into adult eyes. Interestingly, no cells were observed
to attach to the retina or incorporate into established, normal
retinal blood vessels (FIG. 2e). This indicates that the Lin.sup.-
HSC compositions of the present invention do not disrupt a normally
developed vasculature and will not initiate abnormal
vascularization in normally developed retinas.
[0091] In order to determine the relationship between an injected
Lin.sup.- HSC compositions of the present invention and retinal
astrocytes, a transgenic mouse was used, which expressed glial
fibrillary acidic protein (GFAP, a marker of astrocytes) and
promoter-driven green fluorescent protein (GFP). Examination of
retinas of these GFAP-GFP transgenic mice injected with Lin.sup.-
HSC from eGFP transgenic mice demonstrated co-localization of the
injected eGFP EPC and existing astrocytes (FIG. 2f-h, arrows).
Processes of eGFP+Lin.sup.- HSC were observed to conform to the
underlying astrocytic network (arrows, FIG. 2g). Examination of
these eyes demonstrated that the injected, labeled cells only
attached to astrocytes; in P6 mouse retinas, where the retinal
periphery does not yet have endogenous vessels, injected cells were
observed adherent to astrocytes in these not yet vascularized
areas. Surprisingly, injected, labeled cells were observed in the
deeper layers of the retina at the precise location where normal
retinal vessels will subsequently develop (FIG. 2i, arrows).
[0092] To determine whether injected Lin.sup.- HSC of the present
invention are stably incorporated into the developing retinal
vasculature, retinal vessels at several later time points were
examined. As early as P9 (seven days after injection), Lin.sup.-
HSC incorporated into CD31.sup.+ structures (FIG. 2j). By P16 (14
days after injection), the cells were already extensively
incorporated into retinal vascular-like structures (FIG. 2k). When
rhodamine-dextran was injected intravascularly (to identify
functional retinal blood vessels) prior to sacrificing the animals,
the majority of Lin.sup.- HSC were aligned with patent vessels
(FIG. 21). Two patterns of labeled cell distribution were observed:
(1) in one pattern, cells were interspersed along vessels in
between unlabeled endothelial cells; and (2) the other pattern
showed that vessels were composed entirely of labeled cells.
Injected cells were also incorporated into vessels of the deep
vascular plexus (FIG. 2m). While sporadic incorporation of
Lin.sup.- HSC-derived EPC into neovasculature has been previously
reported, this is the first report of vascular networks being
entirely composed of these cells. This demonstrates that cells from
a population of bone marrow-derived Lin.sup.- HSC of the present
invention injected intravitreally can efficiently incorporate into
any layer of the forming retinal vascular plexus.
[0093] Histological examination of non-retinal tissues (e.g.,
brain, liver, heart, lung, bone marrow) did not demonstrate the
presence of any GFP positive cells when examined up to 5 or 10 days
after intravitreal injection. This indicates that a sub-population
of cells within the Lin.sup.- HSC fraction selectively target to
retinal astrocytes and stably incorporate into developing retinal
vasculature. Since these cells have many characteristics of
endothelial cells (association with retinal astrocytes, elongate
morphology, stable incorporation into patent vessels and not
present in extravascular locations), these cells represent EPC
present in the Lin.sup.- HSC population. The targeted astrocytes
are of the same type observed in many of the hypoxic retinopathies.
It is well known that glial cells are a prominent component of
neovascular fronds observed in DR and other forms of retinal
injury. Under conditions of reactive gliosis and ischemia-induced
neovascularization, activated astrocytes proliferate, produce
cytokines, and up-regulate GFAP, similar to that observed during
neonatal retinal vascular template formation in many mammalian
species including humans.
[0094] Lin.sup.- HSC populations of the present invention will
target activated astrocytes in adult mouse eyes as they do in
neonatal eyes, Lin.sup.- HSC cells were injected into adult eyes
with retinas injured by photo-coagulation (FIG. 3a) or needle tip
(FIG. 3b). In both models, a population of cells with prominent
GFAP staining was observed only around the injury site (FIGS. 3a
and b). Cells from injected Lin.sup.- HSC compositions localized to
the injury site and remained specifically associated with
GFAP-positive astrocytes (FIGS. 3a and b). At these sites,
Lin.sup.- HSC cells were also observed to migrate into the deeper
layer of retina at a level similar to that observed during neonatal
formation of the deep retinal vasculature. Uninjured portions of
retina contained no Lin.sup.- HSC cells, identical to that observed
when Lin.sup.- HSC were injected into normal, uninjured adult
retinas (FIG. 2e). These data indicate that Lin.sup.- HSC
compositions can selectively target activated glial cells in
injured adult retinas with gliosis as well as neonatal retinas
undergoing vascularization.
Intravitreally Injected Lin.sup.- HSC Can Rescue and Stabilize
Degenerating Vasculature
[0095] Since intravitreally injected Lin.sup.- HSC compositions
target astrocytes and incorporate into the normal retinal
vasculature, these cells also stabilize degenerating vasculature in
ischemic or degenerative retinal diseases associated with gliosis
and vascular degeneration. The rd/rd mouse is a model for retinal
degeneration that exhibits profound degeneration of photoreceptor
and retinal vascular layers by one month after birth. The retinal
vasculature in these mice develops normally until P16 at which time
the deeper vascular plexus regresses; in most mice the deep and
intermediate plexuses have nearly completely degenerated by
P30.
[0096] To determine whether HSC can rescue the regressing vessels,
Lin.sup.+ or Lin.sup.- HSC (from Balb/c mice) were injected into
rd/rd mice intravitreally at P6. By P33, after injection with
Lin.sup.+ cells, vessels of the deepest retinal layer were nearly
completely absent (FIGS. 4a and b). In contrast, most Lin.sup.-
HSC-injected retinas by P33 had a nearly normal retinal vasculature
with three parallel, well-formed vascular layers (FIGS. 4a and 4d).
Quantification of this effect demonstrated that the average length
of vessels in the deep vascular plexus of Lin injected rd/rd eyes
was nearly three times greater than untreated or Lin.sup.+
cell-treated eyes (FIG. 4e). Surprisingly, injection of a Lin.sup.-
HSC composition derived from rd/rd adult mouse (FVB/N) bone marrow
also rescued degenerating rd/rd neonatal mouse retinal vasculature
(FIG. 40. Degeneration of the vasculature in rd/rd mouse eyes in
observed as early as 2-3 weeks post-natally. Injection of Lin.sup.-
HSC as late as P15 also resulted in partial stabilization of the
degenerating vasculature in the rd/rd mice for at least one month
(FIGS. 4g and 4h).
[0097] A Lin.sup.- HSC composition injected into younger (e.g., P2)
rd/rd mice also incorporated into the developing superficial
vasculature. By P11, these cells were observed to migrate to the
level of the deep vascular plexus and form a pattern identical to
that observed in the wild type outer retinal vascular layer (FIG.
5a). In order to more clearly describe the manner in which cells
from injected Lin.sup.- HSC compositions incorporate into, and
stabilize, degenerating retinal vasculature in the rd/rd mice, a
Lin.sup.- HSC composition derived from Balb/c mice was injected
into Tie-2-GFP FVB mouse eyes. The FVB mice have the rd/rd genotype
and because they express the fusion protein Tie-2-GFP, all
endogenous blood vessels are fluorescent.
[0098] When non-labeled cells from a Lin.sup.- HSC composition are
injected into neonatal Tie-2-GFP FVB eyes and are subsequently
incorporated into the developing vasculature, there should be
non-labeled gaps in the endogenous, Tie-2-GFP labeled vessels that
correspond to the incorporated, non-labeled Lin.sup.- HSC that were
injected. Subsequent staining with another vascular marker (e.g.,
CD-31) then delineates the entire vessel, permitting determination
as to whether non-endogenous endothelial cells are part of the
vasculature. Two months after injection, CD31-positive, Tie-2-GFP
negative, vessels were observed in the retinas of eyes injected
with the Lin.sup.- HSC composition (FIG. 5b). Interestingly, the
majority of rescued vessels contained Tie-2-GFP positive cells
(FIG. 5c). The distribution of pericytes, as determined by staining
for smooth muscle actin, was not changed by Lin.sup.- HSC
injection, regardless of whether there was vascular rescue (FIG.
5d). These data clearly demonstrate that intravitreally injected
Lin.sup.- HSC compositions of the present invention migrate into
the retina, participate in the formation of normal retinal blood
vessels, and stabilize endogenous degenerating vasculature in a
genetically defective mouse.
Inhibition of Retinal Angiogenesis by Transfected Cells from
Lin.sup.- HSC
[0099] The majority of retinal vascular diseases involve abnormal
vascular proliferation rather than degeneration. Transgenic cells
targeted to astrocytes can be used to deliver an anti-angiogenic
protein and inhibit angiogenesis. Cells from Lin.sup.- HSC
compositions were transfected with T2-tryptophanyl-tRNA synthetase
(T2-TrpRS). T2-TrpRS is a 43 kD fragment of TrpRS that potently
inhibits retinal angiogenesis (FIG. 6a). On P12, retinas of eyes
injected with a control plasmid-transfected Lin.sup.- HSC
composition (no T2-TrpRS gene) on P2 had normal primary (FIG. 6c)
and secondary (FIG. 6d) retinal vascular plexuses. When the
T2-TrpRS transfected Lin.sup.- HSC composition of the present
invention was injected into P2 eyes and evaluated 10 days later,
the primary network had significant abnormalities (FIG. 6e) and
formation of the deep retinal vasculature was nearly completely
inhibited (FIG. 6f). The few vessels observed in these eyes were
markedly attenuated with large gaps between vessels. The extent of
inhibition by T2-TrpRS-secreting Lin.sup.- HSCs is detailed in
Table 2.
[0100] T2-TrpRS is produced and secreted by cells in the Lin.sup.-
HSC composition in vitro and after injection of these transfected
cells into the vitreous, a 30 kD fragment of T2-TrpRS in the retina
(FIG. 6b) was observed. This 30 kD fragment was specifically
observed only in retinas injected with transfected Lin.sup.- HSC of
the present invention and this decrease in apparent molecular
weight compared to the recombinant or in vitro-synthesized protein
may be due to processing or degradation of the T2-TrpRS in vivo.
These data indicate that Lin.sup.- HSC compositions can be used to
deliver functionally active genes, such as genes expressing
angiostatic molecules, to the retinal vasculature by targeting to
activated astrocytes. While it is possible that the observed
angiostatic effect is due to cell-mediated activity this is very
unlikely since eyes treated with identical, but non-T2-transfected
Lin.sup.- HSC compositions had normal retinal vasculature.
TABLE-US-00002 TABLE 2 Vascular Inhibition by T2-TrpRS-secreting
Lin.sup.- HSCs Primary Plexus Deep Plexus Inhibited Normal Complete
Partial Normal TsTrpRs 60% 40% 33.3% 60% 6.7% (15 eyes) (9 eyes) (6
eyes) (5 eyes) (9 eyes) (1 eye) Control 0% 100% 0% 38.5% 61.5% (13
eyes) (0 eyes) (13 eyes) (0 eyes) (5 eyes) (8 eyes)
[0101] Intravitreally injected Lin.sup.- HSC populations localize
to retinal astrocytes, incorporate into vessels, and can be useful
in treating many retinal diseases. While most cells from injected
HSC compositions adhere to the astrocytic template, small numbers
migrate deep into the retina, homing to regions where the deep
vascular network will subsequently develop. Even though no
GFAP-positive astrocytes were observed in this area prior to 42
days postnatally, this does not rule out the possibility that
GFAP-negative glial cells are already present to provide a signal
for Lin.sup.- HSC localization. Previous studies have shown that
many diseases are associated with reactive gliosis. In DR, in
particular, glial cells and their extracellular matrix are
associated with pathological angiogenesis.
[0102] Since cells from injected Lin.sup.- HSC compositions
specifically attached to GFAP-expressing glial cells, regardless of
the type of injury, Lin.sup.- HSC compositions of the present
invention can be used to target pre-angiogenic lesions in the
retina. For example, in the ischemic retinopathies such as
diabetes, neovascularization is a response to hypoxia. By targeting
Lin.sup.- HSC compositions to sites of pathological
neovascularization, developing neovasculature can be stabilized
preventing abnormalities of neovasculature such as hemorrhage or
edema (the causes of vision loss associated with DR) and can
potentially alleviate the hypoxia that originally stimulated the
neovascularization. Abnormal blood vessels can be restored to
normal condition. Furthermore, angiostatic proteins, such as
T2-TrpRS can be delivered to sites of pathological angiogenesis by
using transfected Lin.sup.- HSC compositions and laser-induced
activation of astrocytes. Since laser photocoagulation is a
commonly used in clinical ophthalmology, this approach has
application for many retinal diseases. While such cell-based
approaches have been explored in cancer therapy, their use for eye
diseases is more advantageous since intraocular injection makes it
possible to deliver large numbers of cells directly to the site of
disease.
Neurotrophic and Vasculotrophic Rescue by Lin.sup.- HSC
[0103] MACS was used to separate Lin.sup.- HSC from bone marrow of
enhanced green fluorescent protein (eGFP), C3H (rd/rd), FVB (rd/rd)
mice as described above. Lin.sup.- HSC containing EPC from these
mice were injected intravitreally into P6 C3H or FVB mouse eyes.
The retinas were collected at various time points (1 month, 2
months, and 6 months) after injection. The vasculature was analyzed
by scanning laser confocal microscope after staining with
antibodies to CD31 and retinal histology after nuclear staining
with DAPI. Microarray gene expression analysis of mRNA from retinas
at varying time points was also used to identify genes potentially
involved in the effect.
[0104] Eyes of rd/rd mice had profound degeneration of both
neurosensory retina and retinal vasculature by P21. Eyes of rd/rd
mice treated with Lin.sup.- HSC on P6 maintained a normal retinal
vasculature for as long as 6 months; both deep and intermediate
layers were significantly improved when compared to the controls at
all timepoints (1M, 2M, and 6M) (see FIG. 12). In addition, we
observed that retinas treated with Lin.sup.- HSC were also thicker
(1M; 1.2-fold, 2M; 1.3-fold, 6M; 1.4-fold) and had greater numbers
of cells in the outer nuclear layer (1 M; 2.2-fold, 2M; 3.7-fold,
6M; 5.7-fold) relative to eyes treated with Lin.sup.+ HSC as a
control. Large scale genomic analysis of `rescued` (e.g., Lin.sup.-
HSC) compared to control (untreated or non-Lin.sup.- treated) rd/rd
retinas demonstrated a significant upregulation of genes encoding
sHSPs (small heat shock proteins) and specific growth factors that
correlated with vascular and neural rescue, including genes
encoding the proteins listed in FIG. 20, panels A and B.
[0105] The bone marrow derived Lin.sup.- HSC populations of the
present invention significantly and reproducibly induced
maintenance of a normal vasculature and dramatically increased
photoreceptor and other neuronal cell layers in the rd/rd mouse.
This neurotrophic rescue effect correlated with significant
upregulation of small heat shock proteins and growth factors and
provides insights into therapeutic approaches to currently
untreatable retinal degenerative disorders.
Rd1/rd1 Mouse Retinas Exhibit Profound Vascular and Neuronal
Degeneration.
[0106] Normal postnatal retinal vascular and neuronal development
in mice has been well described and is analogous to changes
observed in the third trimester human fetus (Dorrell et al., 2002,
Invest. Ophthalmol. Vis. Sci. 43:3500-3510). Mice homozygous for
the rd1 gene share many characteristics of human retinal
degeneration (Frasson et al., 1999, Nat. Med. 5:1183-1187) and
exhibit rapid photoreceptor (PR) loss accompanied by severe
vascular atrophy as the result of a mutation in the gene encoding
PR cGMP phosphodiesterase (Bowes et al. 1990, Nature 347:677-680).
To examine the vasculature during retinal development and its
subsequent degeneration, antibodies against collagen IV (CIV), an
extracellular matrix (ECM) protein of mature vasculature, and CD31
(PECAM-1), a marker for endothelial cells were used (FIG. 15).
Retinas of rd1/rd1 (C3H/HeJ) developed normally until approximately
postnatal day (P) 8 when degeneration of the
photoreceptor-containing outer nuclear layer (ONL) began. The ONL
rapidly degenerated and cells died by apoptosis such that only a
single layer of nuclei remained by P20. Double staining of the
whole-mounted retinas with antibodies to both CIV and CD31 revealed
details of the vascular degeneration in rd1/rd1 mice similar to
that described by others (Blanks et al., 1986, J. Comp. Neurol.
254:543-553). The primary and deep retinal vascular layers appeared
to develop normally though P12 after which there is a rapid loss of
endothelial cells as evidenced by the absence of CD31 staining.
CD31 positive endothelial cells were present in a normal
distribution through P12 but rapidly disappeared after that.
Interestingly, CIV positive staining remained present throughout
the time points examined, suggesting that the vessels and
associated ECM formed normally, but only the matrix remained after
P13 by which time no CD31 positive cells were observed. (FIG. 15,
middle panels). The intermediate vascular plexus also degenerates
after P21, but the progression is slower than that observed in the
deep plexus (FIG. 15, upper panel). Retinal vascular and neural
cell layers of a normal mouse are shown for comparison to the
rd1/rd1 mouse (right panels, FIG. 15).
Neuroprotective Effect of Bone Marrow-Derived Lin.sup.- HSCs in
rd1/rd1 Mice.
[0107] Intravitreally injected Lin.sup.- HSCs incorporate into
endogenous retinal vasculature in all three vascular plexuses and
prevent the vessels from degenerating. Interestingly, the injected
cells are virtually never observed in the outer nuclear layer.
These cells either incorporate into the forming retinal vessels or
are observed in close proximity to these vessels. Murine Lin.sup.-
HSCs (from C3H/HeJ) were intravitreally injected into C3H/HeJ
(rd1/rd1) mouse eyes at P6, just prior to the onset of
degeneration. By P30, control cell (CD31.sup.-)-injected eyes
exhibited the typical rd1/rd1 phenotype, i.e., nearly complete
degeneration of the deep vascular plexus and ONL was observed in
every retina examined. Eyes injected with Lin.sup.- HSCs maintained
normal-appearing intermediate and deep vascular plexuses.
Surprisingly, significantly more cells were observed in the
internuclear layer (INL) and ONL of Lin.sup.- HSC-injected eyes
than in control cell-injected eyes (FIG. 16A). This rescue effect
of Lin.sup.- HSCs could be observed at 2 months (FIG. 16B) and for
as long as 6 months after injection (FIG. 16C). Differences in the
vasculature of the intermediate and deep plexuses of Lin.sup.-
HSC-injected eyes, as well as the neuronal cell-containing INL and
ONL, were significant at all time points measured when rescued and
non-rescued eyes were compared (FIGS. 16B and C). This effect was
quantified by measuring the total length of the vasculature (FIG.
16D) and counting the number of DAPI-positive cell nuclei observed
in the ONL (FIG. 16E). Simple linear-regression analysis was
applied to the data at all time points.
[0108] A statistically significant correlation was observed between
vascular rescue and neuronal (e.g., ONL thickness) rescue at P30
(p<0.024) and P60 (p<0.034) in the Lin.sup.- HSC-injected
eyes (FIG. 16F). The correlation remained high, although not
statistically significant (p<0.14) at P180 when comparing
Lin.sup.- HSC-injected retinas to control cell-injected retinas
(FIG. 16F). In contrast, control cell-injected retinas showed no
significant correlation between the preservation of vasculature and
ONL at any time point (FIG. 16F). These data demonstrate that
intravitreal injection of Lin.sup.- HSCs results in concomitant
retinal vascular and neuronal rescue in retinas of rd1/rd1 mice.
Injected cells were not observed in the ONL or any place other than
within, or in close proximity to, retinal blood vessels.
Functional Rescue of Lin.sup.- HSC-Injected rd/rd Retinas
[0109] Electroretinograms (ERGs) were performed on mice 2 months
after injection of control cells or murine Lin.sup.- HSCs (FIG.
17). Immunohistochemical and microscopic analysis was done with
each eye following ERG recordings to confirm that vascular and
neuronal rescue had occurred. Representative ERG recordings from
treated, rescued and control, non-rescued eyes show that in the
rescued eyes, the digitally subtracted signal (treated minus
untreated eyes) produced a clearly detectable signal with an
amplitude on the order of 8-10 microvolts (FIG. 17). Clearly, the
signals from both eyes are severely abnormal. However, consistent
and detectable ERGs were recordable from the Lin.sup.- HSC-treated
eyes. In all cases the ERG from the control eye was non-detectable.
While the amplitudes of the signals in rescued eyes were
considerably lower than normal, the signals were consistently
observed whenever there was histological rescue and were on the
order of magnitude of those reported by other, gene based, rescue
studies. Overall these results are demonstrate of some degree of
functional rescue in the eyes treated with the Lin.sup.- HSCs of
the invention.
Human Bone Marrow (hBM)-Derived Lin.sup.- HSCs Also Rescue
Degenerating Retinas
[0110] Lin.sup.- HSCs isolated from human bone marrow behave
similarly to murine Lin.sup.- HSCs. Bone marrow was collected from
human donors and the Lin.sup.+ HSCs were depleted, producing a
population of human Lin.sup.- HSCs (hLin.sup.- HSCs). These cells
were labeled ex-vivo with fluorescent dye and injected into
C3SnSmn.CB17-Prkdc SCID mouse eyes. The injected hLin.sup.- HSCs
migrated to, and targeted, sites of retinal angiogenesis in a
fashion identical to that observed when murine Lin.sup.- HSCs were
injected (FIG. 18A). In addition to the vascular targeting, the
human Lin.sup.- HSCs also provided a robust rescue effect on both
the vascular and neuronal cell layers of the rd1/rd1 mice (FIGS.
18B and 18C). This observation confirms the presence of cells in
human bone marrow that target retinal vasculature and can prevent
retinal degeneration.
Lin.sup.- HSCs have Vasculo- and Neurotrophic Effects in the
rd10/rd10 Mouse
[0111] While the rd1/rd1 mouse is the most widely used and best
characterized model for retinal degeneration (Chang et al. 2002,
Vision Res. 42:517-525), the degeneration is very rapid and in this
regard differs from the usual, slower time course observed in the
human disease. In this strain, photoreceptor cell degeneration
begins around P8, a time when the retinal vasculature is still
rapidly expanding (FIG. 15). Subsequent degeneration of the deep
retinal vasculature occurs even while the intermediate plexus is
still forming and, thus, the retinas of rd1/rd1 mice never
completely develops, unlike that observed in most humans with this
disease. An rd10 mouse model, which has a slower time course of
degeneration and more closely resembles the human retinal
degenerative condition, was used to investigate Lin.sup.-
HSC-mediated vascular rescue. In the rd10 mouse, photoreceptor cell
degeneration begins around P21 and vascular degeneration begins
shortly thereafter.
[0112] Since normal neurosensory retinal development is largely
complete by P21, the degeneration is observed to start after the
retina has completed differentiation and in this way is more
analogous to human retinal degenerations than the rd1/rd1 mouse
model. Lin.sup.- HSCs or control cells from rd10 mice were injected
into P6 eyes and the retinas were evaluated at varying time points.
At P21 the retinas from both Lin.sup.- HSC and control
cell-injected eyes appeared normal with complete development of all
vascular layers and normal development of the INL and ONL (FIGS.
18D and 18H). At approximately P21 the retinal degeneration began
and progressed with age. By P30, the control cell-injected retinas
exhibited severe vascular and neuronal degeneration (FIG. 18I),
while the Lin.sup.- HSC-injected retinas maintained nearly normal
vascular layers and photoreceptor cells (FIG. 18E). The difference
between the rescued and non-rescued eyes was more pronounced at
later time points (compare FIGS. 18F and 18G to 18J and 18K). In
the control treated eyes, the progression of vascular degeneration
was very clearly observed by immunohistochemical staining for CD31
and collagen IV (FIG. 18I-K). The control-treated eyes were nearly
completely negative for CD31, whereas collagen IV-positive vascular
"tracks" remained evident, indicating that vascular regression,
rather than incomplete vascular formation, had occurred. In
contrast, Lin.sup.- HSC-treated eyes had both CD31 and collagen
IV-positive vessels that appeared very similar to normal, wild-type
eyes (compare FIGS. 18F and 18I).
Gene Expression Analysis of rd/rd Mouse Retinas after Lin.sup.- HSC
Treatment
[0113] Large scale genomics (microarray analysis) was used to
analyze rescued and non-rescued retinas to identify putative
mediators of neurotrophic rescue. Gene expression in rd1/rd1 mouse
retinas treated with Lin.sup.- HSCs was compared to uninjected
retinas as well as retinas injected with control cells
(CD31.sup.-). These comparisons were each done in triplicate. To be
considered present, genes were required to have expression levels
at least 2-fold higher than background levels in all three
triplicates. Genes that were upregulated 3-fold in Lin.sup.-
HSC-protected retinas compared to control cell-injected and
non-injected rd/rd mouse retinas are shown in FIG. 20, panels A and
B. Many of the significantly upregulated genes, including MAD and
Ying Yang-1 (YY-1), encode proteins with functions involving the
protection of cells from apoptosis. A number of crystallin genes,
which have sequence homology and similar functions to known
heat-shock proteins involving protection of cells from stress, were
also upregulated by Lin.sup.- HSC treated retinas. Expression of
.alpha.-crystallin was localized to the ONL by immunohistochemical
analysis (FIG. 19).
[0114] Messenger RNA from rd1/rd1 mouse retinas rescued with human
Lin.sup.- HSCs were hybridized to human specific Affymetrix U133A
microarray chips. After stringent analysis, a number of genes were
found whose mRNA expression was human specific, above background,
and significantly higher in the human Lin.sup.- HSC rescued retinas
compared to the murine Lin.sup.- HSC rescued retinas and the human
control cell-injected non-rescued retinas (FIG. 20, panel C). CD6,
a cell adhesion molecule expressed at the surface of primitive and
newly differentiated CD34+ hematopoietic stem cells, and interferon
alpha 13, another gene expressed by hematopoietic stem cells, were
both found by the microarray bioinformatics technique, validating
the evaluation protocol. In addition, several growth factors and
neurotrophic factors were expressed above background by human
Lin.sup.- HSC rescued mouse retina samples (FIG. 20, panel D).
DISCUSSION
[0115] Markers for lineage-committed hematopoietic cells were used
to negatively select a population of bone marrow-derived Lin.sup.-
HSC containing EPC. While the sub-population of bone marrow-derived
Lin.sup.- HSC that can serve as EPC is not characterized by
commonly used cell surface markers, the behavior of these cells in
developing or injured retinal vasculature is entirely different
than that observed for Lin.sup.+ or adult endothelial cell
populations. These cells selectively target to sites of retinal
angiogenesis and participate in the formation of patent blood
vessels.
[0116] Inherited retinal degenerative diseases are often
accompanied by loss of retinal vasculature. Effective treatment of
such diseases requires restoration of function as well as
maintenance of complex tissue architecture. While several recent
studies have explored the use of cell-based delivery of trophic
factors or stem cells themselves, some combination of both may be
necessary. For example, use of growth factor therapy to treat
retinal degenerative disease resulted in unregulated overgrowth of
blood vessels resulting in severe disruption of the normal retinal
tissue architecture. The use of neural or retinal stem cells to
treat retinal degenerative disease may reconstitute neuronal
function, but a functional vasculature will also be necessary to
maintain retinal functional integrity. Incorporation of cells from
a Lin.sup.- HSCs of the present invention into the retinal vessels
of rd/rd mice stabilized the degenerative vasculature without
disrupting retinal structure. This rescue effect was also observed
when the cells were injected into P15 rd/rd mice. Since vascular
degeneration begins on P16 in rd/rd mice, this observation expands
the therapeutic window for effective Lin.sup.- HSC treatment.
Retinal neurons and photoreceptors are preserved and visual
function is maintained in eyes injected with the Lin.sup.- HSC of
the present invention.
[0117] Adult bone marrow-derived Lin.sup.- HSCs exert profound
vasculo- and neurotrophic effects when injected intravitreally into
mice with retinal degenerative disease. This rescue effect persists
for up to 6 months after treatment and is most efficacious when the
Lin.sup.- HSCs are injected prior to complete retinal degeneration
(up to 16 days after birth in mice that ordinarily exhibit complete
retinal degeneration by 30 days postnatally). This rescue is
observed in 2 mouse models of retinal degeneration and, remarkably,
can be accomplished with adult human bone marrow-derived HSCs when
the recipient is an immunodeficient rodent with retinal
degeneration (e.g., the SCID mouse) or when the donor is a mouse
with retinal degeneration. While several recent reports have
described a partial phenotypic rescue in mice or dogs with retinal
degeneration after viral based gene rescue with the wild type gene
(Ali, et al. 2000, Nat Genet. 25:306-310; Takahashi et al. 1999, J.
Virol. 73:7812-7816; Acland et al. 2001, Nat. Genet. 28:92-95.),
the present invention is the first generic cell-based therapeutic
effect achieved by vascular rescue. Thus, the potential utility of
such an approach in treating a group of diseases (e.g., retinitis
pigmentosa) with over 100 known associated mutations is more
practical than creating individual gene therapies to treat each
known mutation.
[0118] The precise molecular basis of the neurotrophic rescue
effect remains unknown, but is observed only when there is
concomitant vascular stabilization/rescue. The presence of injected
stem cells, per se, is not sufficient to generate a neurotrophic
rescue and the clear absence of stem cell-derived neurons in the
outer nuclear layer rules out the possibility that the injected
cells are transforming into photoreceptors. Data obtained by
microarray gene expression analysis demonstrated a significant
up-regulation of genes known to have anti-apoptotic effects. Since
most neuronal death observed in retinal degenerations is by
apoptosis, such protection may be of great therapeutic benefit in
prolonging the life of photoreceptors and other neurons critical to
visual function in these diseases. C-myc is a transcription factor
that participates in apoptosis by upregulation various downstream
apoptosis-inducing factors. C-myc expression was increased 4.5 fold
in rd/rd mice over wild-type indicating potential involvement in
the photoreceptor degeneration observed in the rd1/rd1 mouse. Mad1
and YY-1, two genes dramatically upregulated in Lin.sup.-
HSC-protected retinas (FIG. 20, panel A), are known to suppress the
activity of c-myc, thus inhibiting c-myc induced apoptosis.
Overexpression of Mad1 has also been shown to suppress Fas-induced
activation of caspase-8, another critical component of the
apoptotic pathway. Upregulation of these two molecules may play a
role in protection of the retina from vascular and neural
degeneration by preventing the initiation of apoptosis that
normally leads to degeneration in rd/rd mice.
[0119] Another set of genes that were greatly upregulated in
Lin.sup.- HSC protected retinas includes members of the crystallin
family (FIG. 20, panel B). Similar to heat-shock and other
stress-induced proteins, crystallins may be activated by retinal
stress and provide a protective effect against apoptosis.
Abnormally low expression of .alpha.A-crystallins is correlated
with photoreceptor loss in a rat model of retinal dystrophy and a
recent proteomic analysis of the retina in the rd/rd mouse
demonstrated induction of crystalline up-regulation in response to
retinal degeneration. Based on our microarray data of EPC-rescued
rd/rd mouse retinas, upregulation of crystallins appear to play a
key role in EPC mediated retinal neuroprotection.
[0120] Genes such as c-myc, Mad1, Yx-1 and the crystallins are
likely to be downstream mediators of neuronal rescue. Neurotrophic
agents can regulate anti-apoptotic gene expression, although our
microarray analysis of retinas rescued with mouse stem cells did
not demonstrate induction of increased levels of known neurotrophic
factors. Analysis of human bone marrow-derived stem cell-mediated
rescue with human specific chips did, on the other hand,
demonstrate low, but significant increases in the expression of
multiple growth factor genes.
[0121] The upregulated genes include several members of the
fibroblast growth factor family and otoferlin. Mutations in the
otoferlin gene are associated with genetic disorders leading to
deafness due to auditory neuropathy. It is possible that otoferlin
production by injected Lin.sup.- HSCs contributes to the prevention
of retinal neuropathy as well. Historically, it has long been
assumed that vascular changes observed in patients and animals with
retinal degeneration were secondary to decreased metabolic demand
as the photoreceptors die. The present data indicate that, at least
for mice with inherited retinal degeneration, preserving normal
vasculature can help maintain components of the outer nuclear layer
as well. Recent reports in the literature would support the concept
that tissue-specific vasculature has trophic effects that go beyond
that expected from simply providing vascular "nourishment." For
example, liver endothelial cells can be induced to produce, after
VEGFR1 activation, growth factors critical to hepatocyte
regeneration and maintenance in the face of hepatic injury
(LeCouter et al. 2003, Science 299:890-893).
[0122] Similar indicative interactions between vascular endothelial
cells and adjacent hepatic parenchymal cells are reportedly
involved in liver organogenesis, well before the formation of
functional blood vessels. Endogenous retinal vasculature in
individuals with retinal degeneration may not facilitate so
dramatic a rescue, but if this vasculature is buttressed with
endothelial progenitors derived from bone marrow hematopoietic stem
cell populations, they may make the vasculature more resistant to
degeneration and at the same time facilitate retinal neuronal, as
well as vascular, survival. In humans with retinal degeneration,
delaying the onset of complete retinal degeneration may provide
years of additional sight. The animals treated with the Lin.sup.-
HSCs of the present invention had significant preservation of an
ERG, which may be sufficient to support vision.
[0123] Clinically, it is widely appreciated that there can be
substantial loss of photoreceptors and other neurons while still
preserving functional vision. At some point, the critical threshold
is crossed and vision is lost. Since nearly all of the human
inherited retinal degenerations are of early, but slow, onset, it
may be possible to identify an individual with retinal
degeneration, treat them intravitreally with an autologous bone
marrow stem cell graft and delay retinal degeneration with
concomitant loss of vision. To enhance targeting and incorporation
of these stem cells, the presence of activated astrocytes would be
desirable. This can be accomplished by early treatment when there
is an associated gliosis or by using a laser to stimulate local
proliferation of activated astrocytes.
[0124] The Lin.sup.- HSC populations of the present invention
contain a population of EPC that can promote angiogenesis by
targeting reactive astrocytes and incorporate into an established
template without disrupting retinal structure. The Lin.sup.- HSC of
the present invention also provide a surprising long-term
neurotrophic rescue effect in eyes suffering from retinal
degeneration. In addition, genetically modified, autologous
Lin.sup.- HSC compositions containing EPC can be transplanted into
ischemic or abnormally vascularized eyes and can stably incorporate
into new vessels and continuously deliver therapeutic molecules
locally for prolonged periods of time. Such local delivery of genes
that express pharmacological agents in physiologically meaningful
doses represents a new paradigm for treating currently untreatable
ocular diseases.
[0125] Numerous variations and modifications of the embodiments
described above may be effected without departing from the spirit
and scope of the novel features of the invention. No limitations
with respect to the specific embodiments illustrated herein are
intended or should be inferred.
Sequence CWU 1
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gcctgtggct ttgacatcaa 3840caagactttc atattctctg acctggacta
catggggatg agctcaggtt tctacaaaaa 3900tgtggtgaag attcaaaagc
atgttacctt caaccaagtg aaaggcattt tcggcttcac 3960tgacagcgac
tgcattggga agatcagttt tcctgccatc caggctgctc cctccttcag
4020caactcattc ccacagatct tccgagacag gacggatatc cagtgcctta
tcccatgtgc 4080cattgaccag gatccttact ttagaatgac aagggacgtc
gcccccagga tcggctatcc 4140taaaccagcc ctgttgcact ccaccttctt
cccagccctg cagggcgccc agaccaaaat 4200gagtgccagc gacccaaact
cctccatctt cctcaccgac acggccaagc agatcaaaac 4260caaggtcaat
aagcatgcgt tttctggagg gagagacacc atcgaggagc acaggcagtt
4320tgggggcaac tgtgatgtgg acgtgtcttt catgtacctg accttcttcc
tcgaggacga 4380cgacaagctc gagcagatca ggaaggatta caccagcgga
gccatgctca ccggtgagct 4440caagaaggca ctcatagagg ttctgcagcc
cttgatcgca gagcaccagg cccggcgcaa 4500ggaggtcacg gatgagatag
tgaaagagtt catgactccc cggaagctgt ccttcgactt 4560tcagaagctt
gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa
4620caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
tagcataacc 4680ccttggggcc tctaaacggg tcttgagggg ttttttgctg
aaaggaggaa ctatatccgg 4740at 47422392PRTArtificial
SequenceHis-tagged human T2-TrpRS 2Met Ser Ala Lys Gly Ile Asp Tyr
Asp Lys Leu Ile Val Arg Phe Gly1 5 10 15Ser Ser Lys Ile Asp Lys Glu
Leu Ile Asn Arg Ile Glu Arg Ala Thr 20 25 30Gly Gln Arg Pro His His
Phe Leu Arg Arg Gly Ile Phe Phe Ser His 35 40 45Arg Asp Met Asn Gln
Val Leu Asp Ala Tyr Glu Asn Lys Lys Pro Phe 50 55 60Tyr Leu Tyr Thr
Gly Arg Gly Pro Ser Ser Glu Ala Met His Val Gly65 70 75 80His Leu
Ile Pro Phe Ile Phe Thr Lys Trp Leu Gln Asp Val Phe Asn 85 90 95Val
Pro Leu Val Ile Gln Met Thr Asp Asp Glu Lys Tyr Leu Trp Lys 100 105
110Asp Leu Thr Leu Asp Gln Ala Tyr Gly Asp Ala Val Glu Asn Ala Lys
115 120 125Asp Ile Ile Ala Cys Gly Phe Asp Ile Asn Lys Thr Phe Ile
Phe Ser 130 135 140Asp Leu Asp Tyr Met Gly Met Ser Ser Gly Phe Tyr
Lys Asn Val Val145 150 155 160Lys Ile Gln Lys His Val Thr Phe Asn
Gln Val Lys Gly Ile Phe Gly 165 170 175Phe Thr Asp Ser Asp Cys Ile
Gly Lys Ile Ser Phe Pro Ala Ile Gln 180 185 190Ala Ala Pro Ser Phe
Ser Asn Ser Phe Pro Gln Ile Phe Arg Asp Arg 195 200 205Thr Asp Ile
Gln Cys Leu Ile Pro Cys Ala Ile Asp Gln Asp Pro Tyr 210 215 220Phe
Arg Met Thr Arg Asp Val Ala Pro Arg Ile Gly Tyr Pro Lys Pro225 230
235 240Ala Leu Leu His Ser Thr Phe Phe Pro Ala Leu Gln Gly Ala Gln
Thr 245 250 255Lys Met Ser Ala Ser Asp Pro Asn Ser Ser Ile Phe Leu
Thr Asp Thr 260 265 270Ala Lys Gln Ile Lys Thr Lys Val Asn Lys His
Ala Phe Ser Gly Gly 275 280 285Arg Asp Thr Ile Glu Glu His Arg Gln
Phe Gly Gly Asn Cys Asp Val 290 295 300Asp Val Ser Phe Met Tyr Leu
Thr Phe Phe Leu Glu Asp Asp Asp Lys305 310 315 320Leu Glu Gln Ile
Arg Lys Asp Tyr Thr Ser Gly Ala Met Leu Thr Gly 325 330 335Glu Leu
Lys Lys Ala Leu Ile Glu Val Leu Gln Pro Leu Ile Ala Glu 340 345
350His Gln Ala Arg Arg Lys Glu Val Thr Asp Glu Ile Val Lys Glu Phe
355 360 365Met Thr Pro Arg Lys Leu Ser Phe Asp Phe Gln Lys Leu Ala
Ala Ala 370 375 380Leu Glu His His His His His His385 390
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