KR101309500B1 - Isolated lineage negative hematopoietic stem cells and methods of treatment therewith - Google Patents

Abstract

The isolated, lineage negative hematopoietic stem cells (Lin HSCs) from the adult adult bone marrow contain endothelial progenitor cells (EPCs) that can rescue retinal blood vessels and neuronal networks in the eye. Preferably, at least about 20% of the cells in the isolated Lin HSCs express the cell surface antigen CD31. The isolated Lin HSC populations are useful for the treatment of ocular vascular diseases and for improving cortical cell degeneration in the retina. In a preferred embodiment, Lin HSC extracts bone marrow from adult mammals; Separating a number of monocytes from the bone marrow; Monocytes are labeled with biotin-conjugated line panel antibody to one or more lineage surface antigens; Removing monocytes positive for lineage surface antigen from a plurality of said monocytes; Isolates by recovering Lin HSC populations containing EPC. Isolated Lin HSCs can also be transfected with therapeutically useful genes. The treatment can be enhanced by stimulating the proliferation of activated astrocytes in the retina using a laser.

Lineage negative hematopoietic stem cells, endothelial progenitor cells, vertebral cells, retinal degeneration, surface antigen

Description

Isolated lineage negative hematopoietic stem cells and methods of treatment therewith

Cross reference of related application

This application is filed on July 25, 2003, which claims the benefit of Provisional Patent Application No. 60 / 398,522 filed July 25, 2002 and Provisional Patent Application No. 60 / 467,051 filed May 2, 2003. The CIP application of US patent application Ser. No. 10 / 833,743, filed April 28, 2004, the CIP application of US patent application Ser. No. 10 / 628,783, which is incorporated by reference in its entirety.

Statement of Government Rights

Some of the studies described herein are provided by the aid number CA92577 from the National Cancer Institute and by the aid numbers EY11254, EY12598 and EY125998 from the National Institute of Health. Supported. The United States Government has certain rights in the invention.

The present invention relates to an isolated, mammalian stem cell. More specifically, the present invention treats the eye of a mammal with a lineage negative hematopoietic stem cell (Lin ^- HSC) population derived from bone marrow, and a separate Lin ^- HSC population, and is suffering from ocular degeneration. A method for preserving cone cells in the retina of a mammal.

Aging-related macular degeneration (ARMD) and diabetic retinopathy (DR) are the leading cause of vision loss in industrialized countries and occur as a result of abnormal neovascularization. Because the retina consists of very clear layers of neuronal, glial and vascular elements, even relatively small disorders, as seen in vascular proliferation or edema, can cause significant loss of visual function. Hereditary retinal degeneration, such as retinal pigment degeneration (RP), is also associated with vascular abnormalities such as arterioles narrowing and vascular atrophy. Most hereditary human retinal degenerations specifically affect hepatic photoreceptors, but are accompanied by loss of the vertebrae, a major cellular component of the macula, the region of the human retina responsible for central, microscopic vision. Cone-specific survival factors have recently been reported (MohandSaid et al . 1998, Proc. Natl. Acad. Sci. USA, 95: 8357-8362), which can promote vertebral survival in mouse models of retinal degeneration.

Hereditary retinal degeneration is the majority of 1 in 3500 people and is characterized by progressive night blindness, blindness, optic nerve atrophy, arteriosclerosis, vascular permeability alteration, and central vision loss, which often progresses to complete blindness. Heckenlively, JR, editor, 1988; Retinitis Pigmentosa, Philadelphia; JB Lippincott Co.]. Molecular genetic analysis of these diseases has identified mutations in at least 110 different genes that are responsible for only a relatively small proportion of known diseases. Humphries et al., 1992, Science 256: 804-808; Farrar et al., 2002, EMBO J. 21: 857-864. Many of these mutations relate to the enzyme components and structural components of the photoconversion machinery, including rhodopsin, cGMP phosphodiesterase, rds periprine, and RPE65. Despite these findings, there are still no effective therapies to delay or reverse the progression of such retinal degeneration diseases. Recent advances in gene therapy have resulted in the rds phenotype of mice when wild type transgenes are delivered to photoreceptors or retinal pigment epithelium (RPE) in animals with specific mutations [Ali et al. 2000, Nat. Genet. 25: 306-310 and the rd phenotype (Takahashi et al., 1999, J. Virol. 73: 7812-7816] and the RPE65 phenotype of dogs (Acland et al., 2001, Nat. Genet. 28: 92-95 successfully led the reversal.

For many years, it has been known that stem cell populations exist in the normal adult circulation and bone marrow. Different subpopulations of these cells can differentiate along hematopoietic lineage positive (Lin ⁺ ) or lineage negative (Lin ⁻ ) lineages. In addition, lineage negative hematopoietic stem cell (HSC) populations have recently been found to contain endothelial progenitor cells (EPCs) that can form blood vessels in vitro and in vivo. Asahara et al. 1997, Science 275: 964-7. These cells may be involved in normal and pathological postnatal angiogenesis, as well as in Lyden et al. 2001, Nat. Med. 7: 1194-201; Kalka et al. 2000, Proc. Natl. Acad. Sci. USA 97: 3422-7; and Kocher et al. 2001, Nat. Med. 7: 430-6], hepatocytes [Lagasse et al. 2000, Nat. Med. 6: 1229-34], microglia (Priller et al. 2002, Nat. Med. 7: 1356-61], cardiomyocytes [Orlac et al. 2001, Proc. Natl. Acad. Sci. USA 98: 10344-9] and epithelium [Lyden et al. 2001, Nat. Med. 7: 1194-201] can be differentiated into various non-endothelial cell types. Although these cells have been used in several experimental models of angiogenesis, the mechanism of EPC targeting neovascular system is unknown and no strategy has been identified to effectively increase the number of cells contributing to a particular vascular system.

Bone marrow-derived hematopoietic stem cells are currently the only kind of stem cells commonly used for treatment. Bone marrow HSCs have been used for transplants for over 40 years. Currently, advanced methods of harvesting purified stem cells are being investigated for the development of therapies for treating leukemia, lymphoma and hereditary blood diseases. Clinical applications of human stem cells are being reviewed for the treatment of diabetes and advanced kidney cancer in a limited number of human patients.

Summary of the Invention

The present invention provides a method for improving vertebral cell degeneration in the retina of a mammal suffering from eye disease. The method comprises administering to the mammalian retina a lineage-negative hematopoietic stem cell population comprising a mammalian bone marrow-derived isolated hematopoietic stem cell and endothelial progenitor cell. The cells are administered in an amount sufficient to retard vertebral cell degeneration in the retina.

A preferred method is to isolate a lineage-negative hematopoietic stem cell population comprising endothelial progenitor cells from the bone marrow of a mammal suffering from eye disease, and then isolate the stem cells in sufficient numbers to improve the degeneration of the cortical cells of the retina. Injection into the vitreous into the eye.

The method of the present invention is directed to a population of isolated mammalian lineage negative hematopoietic stem cells (Lin ^- HSC) derived from mammalian bone marrow (ie, hematopoietic stem cells that do not express lineage surface antigen (Lin) on the cell surface) ( HSC)). Preferably, the cells are autologous stem cells (ie, derived from bone marrow of the mammalian subject to be treated). The Lin ^- HSC population of isolated mammals includes endothelial progenitor cells (EPCs), also known as endothelial progenitor cells, which selectively target activated retinal stellate cells when injected intraocularly into the eye. Preferably the mammal is a human.

In a preferred embodiment, the Lin ^- HSC population of the present invention comprises extracting bone marrow from a mammal suffering from an eye disease; Separating the plurality of monocytes from the bone marrow; Labeling the monocytes with a biotin-conjugated line panel antibody against one or more lineage surface antigens; Removing monocytes positive for lineage surface antigen; And recovering the Lin ^- HSC population containing EPC. Preferably, the monocytes are CD2, CD3, CD4, CD11, CD11a, Mac-11, CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, Ly-6G, TER-119, CD45RA, CD56, CD64, Labeled with biotin-conjugated lineage panel antibodies to one or more lineage surface antigens selected from the group consisting of CD68, CD86, CD66b, HLA-DR and CD235a (glycoporin A). Preferably, at least about 20% of the cells of the isolated Lin ^- HSC population of the invention express surface antigen CD31. The isolated cells are then administered to the diseased eye of the mammal, preferably by intraocular injection. In a preferred embodiment, separate Lin ^- HSC is an about 50% or more of the expression of surface antigens CD31 and to separate Lin ^- about 50% or more of the HSC has a surface antigen expression of CD117 (c-kit).

EPCs in the Lin ^- HSC population of the present invention invade extensively developing retinal vessels and retinal neuronal networks, stably incorporating into the ocular neovascular system and neuronal networks. Normal mouse retinas dominated by rods, but in mice treated by the methods of the invention, the cells rescued after treatment with Lin ^- HSC were surprisingly almost all conical.

In one preferred embodiment, cells of the isolated Lin ^- HSC population are transfected with a therapeutically useful gene. For example, the cell selectively targets the neovascular system to operate neurotrophic and anti-angiogenic agents that inhibit new blood vessel formation without affecting blood vessels already established through cell-based gene therapy forms. Possibly transfected with a polynucleotide that encodes. In one aspect, isolated Lin ^- HSC populations useful in the methods of the invention include genes encoding angiogenesis inhibitory peptides. Inhibitory angiogenesis Lin ^- HSC is useful for regulating 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 ^- HSC of the present invention comprises a gene encoding a neurotrophic peptide. The neurotrophic Lin ^- HSC is useful for promoting the rescue of neurons in eye diseases including retinal nerve degeneration, such as glaucoma, retinal pigment degeneration.

A particular advantage of ocular therapies using the isolated Lin ^- HSC population of the present invention is the vasotrophic and neurotrophic rescue effects observed in the eye treated intralinically with Lin ^- HSC. In the eye treated with the isolated Lin ^- HSC of the present invention, retinal neurons and photoreceptors, especially vertebral bodies, can be preserved and visual function maintained to some extent.

Preferably, the retina affected by the disease to be treated by the method of the present invention comprises activated astrocytes. This can be achieved by early treatment of the eye in the presence of associated neuroglia, or by stimulating local proliferation of activated astrocytic cells using a laser.

In the drawings,

1 shows a schematic illustration of the developing mouse retina. 1A: Development of primary vascular plexus. 1B: Second phase of retinal angiogenesis. GCL, ganglion cell layer; IPL, internal plexus layer; INL, inner nuclear layer; OPL, outer plexus layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium; ON, optic nerve; P, periphery.

FIG. 1C depicts flow cytometry characteristics of cells isolated with bone marrow-derived Lin ⁺ HSC and Lin ^- HSC. Top row: dot plot distribution of non-antibody labeled cells, wherein R1 represents the quantifiable-opening area of positive PE-staining and R2 represents GFP-positive; Middle row: Lin ^- HSC (C57B / 6) and bottom row: Lin ⁺ HSC (C57B / 6) cells, each cell line PE-conjugated for Sca-1, c-kit, Flk-1 / KDR, CD31 Labeled with an antibody. Tie-2 data was obtained from Tie-2-GFP mice. % Represents the percentage of positively-labeled cells in the entire Lin ⁻ HSC or Lin ⁺ HSC population.

2 shows the engraftment of Lin ⁻ HSC into the developing mouse retina. Figure 2a: 4 days post injection (P6), eGFP ⁺ Lin ^- HSC cells injected intravitreally attach and differentiate to the retina. Figure 2B: Lin ^- HSC (B6.129S7-Gtrosa26 mouse, stained with β-gal antibody) is established in front of the vascular system itself stained with collagen IV antibody (* indicates the tip of the vascular system). 2C: Most of Lin ⁺ HSC cells (eGFP ⁺ ) did not differentiate at 4 days (P6) after injection. 2D: Mesenteric eGFP ⁺ rat EC 4 days post injection (P6). 2E: Lin ^- HSC (eGFP ⁺ ) injected into adult mouse eyes. Figure 2F: Low magnification of eGFP ⁺ Lin ^- HSC (arrows) that migrate and differentiate along existing astrocytic template in GFAP-GFP transgenic mice. 2G: High magnification of association (arrow) between Lin ⁻ cells (eGFP) and basal stellate cells. 2H: Non-injected GFAP-GFP transgenic control. FIG. 2i: Day 4 post-injection (P6), eGFP ⁺ Lin ^- HSC cells migrate to and differentiate into future deep plexus regions. The left plot captures Lin ^- HSC activity in the retina of the whole mount and the right plot shows the location (arrow) of Lin ^- cells in the retina (top is the vitreous side and the bottom is the sclera side) to be). 2J: Double labeling with α-CD31-PE and α-GFP-alexa 488 antibodies. Seven days after injection, injected Lin ^- HSC (eGFP, red) was incorporated into the vascular system (CD31). Arrowheads indicate incorporated areas. 2K: eGFP ⁺ Lin ⁻ HSC cells form blood vessels 14 days after injection (P17). 2L and 2M: Intracardiac injection of rhodamine-dextran indicates that blood vessels are intact and functional in both primary plexus (1) and deep plexus (m).

FIG. 3 shows that glioma (GFAP) eGFP ⁺ Lin ⁻ HSC cells are induced by laser induced injury (a) and mechanically induced injury (b) (* indicates damaged sites) in the adult retina. It is shown as a stellate cell, the leftmost image). The rightmost image is a high magnification showing close association of Lin ^- HSC with astrocytes. Scale bar = 20 μM.

4 shows that Lin ^- HSC cells rescue the vascular system of retinal degeneration mice. 4A-4D: retina at day 27 post injection (P33), stained with collagen IV; 4A and 4B, retinas injected with Lin ⁺ HSC cells (Balb / c) showed no difference from the vascular system of normal FVB mice; 4C and 4D: Retinas injected with Lin ⁻ HSC (Balb / c) showed a rich vascular network similar to wild type mice; 4A and 4C: Frozen sections of the entire retina stained with DAPI (top side is vitreous side and bottom side sclera side); 4B and 4D: Deep vascular plexus of retinal warm tissue specimens; 4E: Bar graph showing an increase in the degree of vascular distribution of the deep plexus formed in Lin ^- HSC cell-injected retinas (n = 6). The degree of vascularization of the deep retina was quantified by calculating the total length of blood vessels in each image. Mean total length / high magnification field of view (in microns) of blood vessels of Lin ^- HSC, Lin ⁺ HSC or control retina was compared. 4F: Comparison of deep vascular length after injection of Lin ⁻ HSC (R, right eye) cells or Lin ⁺ HSC (L, right eye) cells from rd / rd mice. Results of 6 independent mice are shown (each color represents each mouse). 4G and 4H: Lin ^- HSC cells also rescued the rd / rd-blood system when injected into (Balb / c) P15 eyes. Intermediate and deep vascular plexuses (one month after injection) of the retina injected with Lin ^- HSC (G) or Lin ⁺ HSC (H) cells are shown.

5 shows micrographs of mouse retinal tissue: FIG. 5A, depth of retinal warm tissue specimens (rd / rd mice) on day 5 (P11) following injection of visible eGFP ⁺ Lin ⁻ HSC (grey). 5B and 5C: P60 retinal vascular system of Tie-2-GFP (rd / rd) mice injected with Balb / c Lin ⁻ cells (b) or Lin ⁺ HSC cells (c) at P6. Only endogenous endothelial cells (GFP stained cells) are visible in the left panel of (b) and (c). The middle panels of (b) and (c) are stained with CD31 antibody, the arrows indicate blood vessels stained with CD31 rather than GFP, and the right panels of (b) and (c) show the staining with GFP and CD31. It is shown. 5D: α-SMA staining of Lin ⁻ HSC injected retina (left panel) and control retina (right panel).

6 shows that T2-TrpRS-transfected Lin ^- HSCs inhibit the development of the mouse retinal vascular system. 6A: Schematic depiction of human TrpRS, T2-TrpRS and T2-TrpRS with Igk signal sequences at the amino terminus. 6B: Retinas injected with T2-TrpRS transfected Lin ^- HSC express T2-TrpRS protein in vivo. (1) Lee. Recombinant T2-TrpRS produced in E. coli; (2) Lee. Recombinant T2-TrpRS produced in E. coli; (3) Lee. Recombinant T2-TrpRS produced in E. coli; (4) control retina; (5) retinas injected with Lin ^- HSC + pSecTag2A (vector only); (6) Retina injected with Lin ^- HSC + pKLe135 (Igk-T2-TrpRS in pSecTag). 6A: endogenous TrpRS. 6B: Recombinant T2-TrpRS. 6C: T2-TrpRS of retina injected with Lin ⁻ HSC. 6C-6F: Representative primary (surface) plexus and secondary (deep) plexus of the injected retina 7 days after injection; 6C and 6D: Eyes injected with empty plasmid-transfected Lin ^- HSC developed normally; 6E and 6F: Eyes injected with the majority of T2-TrpRS-transfected Lin ^- HSC showed inhibition of the deep plexus; 6C and 6E: primary (superficial) vascular plexus; 6D and 6F: Secondary (deep) plexus. The faint outline of the blood vessel observed in FIG. 6F is an “bleed” image of the primary vascular network shown in FIG. 6E.

Figure 7 depicts the DNA sequence of His ₆ -tagged T2-TrpRS (SEQ ID NO: 1).

8 shows the amino acid sequence of His ₆ -tagged T2-TrpRS (SEQ ID NO: 2).

9 shows micrographs and retinal potential maps (ERG) of the retina of mice injected with Lin ^- HSC and Lin ⁺ HSC (control) of the present invention in the eye.

Figure 10 Statistics showing the correlation between neuronal rescue (y-axis) and vascular rescue (x-axis) for both medial plexus (Int.) And deep vascular layers of rd / rd mouse eye treated with Lin ^- HSC. The mathematical plot is shown.

FIG. 11 shows statistical plots showing no correlation between neuronal rescue (y-axis) and vascular rescue (x-axis) for rd / rd mouse eyes treated with Lin ⁺ HSC.

12 shows rd / rd mouse eye (black bars) and untreated rd / rd mouse eye treated with Lin ^- HSC at time points 1 month (1M), 2 months (2M) and 6 months (6M) post-injection. Bar graph of vessel length (y-axis) in any relative units (white bar).

FIG. 13 includes three bar graphs of the number of nuclei in the outer neuronal layer (ONR) of rd / rd mice at time points 1 month (1M), 2 months (2M) and 6 months (6M) post injection. It is demonstrated that the number of nuclei was markedly increased in the case of eyes treated with Lin ^- HSC (black bars) compared to control eyes treated with Lin ⁺ HSC (white bars).

FIG. 14 treated the right eye (treated with R, Lin-HSC) with the left eye (L, Lin ⁺ HSC) at time points 1 month (1M), 2 months (2M) and 6 months (6M) (after injection). A plot of the number of nuclei in the outer neuronal layer for individual rd / rd mice, compared to the control eye), is shown, with each line in the given plot comparing the eyes of the individual mice.

FIG. 15 shows changes in retinal vascular system and neurons in rd1 / rd1 (C3H / HeJ, left panel) mice or wild type mice (C57BL / 6, right panel). Retinal vascular system (red: collagen IV, green: CD31) and sections of the same retina (red: DAPI and green: CD31, bottom panel) of the middle (top panel) or deep (middle panel) plexus of the warm tissue specimen retina (GCL : Ganglion cell layer, INL: inner nuclear layer, ONL: outer nuclear layer) (P: days after birth).

16 shows that Lin ^- HSC injection rescues neuronal degeneration in rd1 / rd1 mice. A, B and C are P30 (A), P60 (B), and P180 Lin observed at (C) ^- HSC injected eye (right panels) and contralateral control cell (CD31 ^-) injected eye (left panels) The retinal vascular system of the middle or deep vascular plexus and section is shown. D represents the vasculature of the retinal or Lin ^- HSC injected retina or control cells (CD31 ⁻ ) injected at P30 (left, n = 10), P60 (middle, n = 10) and P180 (right, n = 6). Mean total length (+ or-is the standard error of the mean). The data of the medial and deep plexus were shown respectively (Y axis: relative length of the vascular system). E is the mean of the nuclei in ONL observed in P30 (left, n = 10), P60 (middle, n = 10) or P180 (right, n = 6) in control cells (CD31 ⁻ ) or Lin ⁻ HSC injected retina Numbers are shown (Y axis: relative number of cell nuclei in ONL). F is a straight line between the length of the vascular system (X axis) and the number of cell nuclei in the ONL (Y axis) observed in P30 (left), P60 (middle) and P180 (right) of the retina injected with Lin ^- HSC or control cells. Correlation is shown.

Figure 17 demonstrates that retinal function is rescued by Lin ^- HSC injection. The ERG using (ERG) recording Lin ^- to measure the function of the retinal scanning ^- HSC or control cell (CD31). A and B show representative examples of the retinal and unrelieved retinas rescued 2 months after injection. Retinal sections of Lin ^- HSC injected right eye (A) and CD31 ^- control cells injected left eye (B) of the same animal (green: CD31 stained vascular system, red: DAPI stained nucleus). C shows the ERG results of the same animal presented in A and B.

FIG. 18 shows that a population of human bone marrow cells can rescue retinal degeneration of rd1 mice (A-C). In addition, rescue was also observed in another model of retinal degeneration, rd10 (DK). A shows that human Lin ^- HSC (hLin ^- HSC) labeled with green dye can be injected into the vitreous of C3SnSmn.CB17-Prkdc SCID mice and then differentiate into retinal vascular cells. B and C are the retinal vasculature of the hLin ^- HSC injected eye (B) or contralateral control eye (C) at 1.5 months post injection (left panel; top: middle plexus, bottom: deep plexus) and nerve cells ( Right panel). DK depicts rescue of rd10 mice by Lin ^- HSC (injected in P6). P21 (D: Lin ^- HSC, J: control cells), P30 (E: Lin ^- HSC, I: control cells), P60 (F: Lin ^- HSC, J: control cells) and P105 (G: Lin ^- HSC, Representative retinas observed in K: control cells) are shown (the treated eye and the control eye are from the same animal at each time point). The retinal vascular system (top image of each panel is the medial vascular plexus, and the middle image of each panel is the deep vascular plexus) was stained with CD31 (green) and collagen IV (red). The bottom image of each panel shows a cross-sectional view of the same retina (red: DAPI, green: CD31).

FIG. 19 shows that crystallin αA is upregulated in rescued outer nuclear layer cells after treatment with Lin ⁻ HSC but not contralateral eye treated with control cells. Left panel: IgG control of rescued retina, middle panel: Crystalline αA of rescued retina, right panel; Crystalline αA in the unrelieved retina.

20 is a diagram of genes that are upregulated in rat retinas treated with Lin ^- HSC of the present invention. (A) Gene with 3-fold increase in expression in mouse retina treated with mouse Lin ^- HSC. (B) Crystalline gene upregulated in mouse retina treated with mouse Lin ^- HSC. (C) Genes with a 2-fold increase in expression in mouse retinas treated with human Lin ^- HSC. (D) Neurotrophic or growth factor genes whose expression is upregulated in mouse retinas treated with human Lin ^- HSC.

Figure 21 depicts the distribution of CD31 and integrin α6 surface antigens observed in the CD133 positive (CD133 ⁺ ) and CD133 negative (CD133 ⁻ ) human Lin ⁻ HSC populations of the present invention. The left panel shows the scatter plot of the flow cytometry. Middle and right panels are histograms showing specific antibody expression levels for a cell population. The Y axis represents the number of events and the X axis represents the strength of the signal. Shifting the filled histogram to the right of the coarse (control) histogram indicates an increase in fluorescent signal and expression of the antibody above the background level.

FIG. 22 shows postnatal retinal development of wild-type C57 / B16 mice that develop at normal oxygen levels (normoxia), observed from postnatal P0 to P30 days.

FIG. 23 shows oxygen-induced retinopathy of C57 / B16 mice that develop at high oxygen levels (hyperoxia; 75% oxygen) between P7 and P12 and subsequent normal oxygen (normoxia) between P12 and P17. The model is shown.

FIG. 24 demonstrates vascular rescue observed upon treatment with the Lin ^- HSC population of the invention in an oxygen-induced retinopathy model.

25 shows that after intravitreal injection of LinHSC, the rescued photoreceptors in the outer nucleus (ONL) layer of rd1 mice are predominantly vertebral. A small proportion of photoreceptors in the wild-type mouse retina (top panel) were vertebrae as evidenced by the expression of red / green vertebral opsin (A), with most cells of ONL positive for hepatic specific rhodopsin. The retinal vascular system emits autofluorescence with preimmune serum (C), but the nuclear layer is completely negative for staining by either interstitial or cone-specific opsin. The Rd / rd Maus retina (bottom panel) had a shrunken inner nuclear layer and a nearly complete atrophy ONL, both of which were negative for the vertebral (D) or simplified (panel G) opsin. The control CD31-HSC treated eye is identical to the non-injected rd / rd retina without any staining for the vertebral (E) or hepatic (H) opsin. Lin-HSC treated contralateral eye showed a markedly reduced but presently present ONL, which consisted mainly of vertebral bodies, as evidenced by the positive immune activity against vertebral red / green opsin (I). A small number of simplifieds was also observed (I).

Stem cells are typically identified by the distribution of antigens on the cell surface [detailed descriptions of which are properly cited herein (Stem Cells: Scientific Progress and Future Directions, National Institutes of Health, Office of Science) Policy Report, 2001.6., Appendix E: Stem Cell Markers).

The present invention provides a method of improving vertebral cell degeneration in the retina of a mammal suffering from eye disease. The lineage negative hematopoietic stem cell population, including mammalian bone marrow-derived isolated, hematopoietic stem cells and endothelial progenitor cells, is administered to the mammalian retina, preferably by intravitreal injection. The cells are administered in an amount sufficient to improve vertebral cell degeneration in the retina.

Preferred methods include the isolation of lineage-negative, hematopoietic stem cells from the bone marrow of the mammal to be treated and then administering the cells to the mammal in sufficient numbers to improve degeneration of the vertebral cells in the retina.

The cells may be obtained from a diseased mammal, preferably a mammal at an early stage of eye disease. Alternatively, the cells can be obtained before the disease develops in patients known to have genetic predisposition to eye diseases such as, for example, retinal pigmentation. The cells can be stored until needed and injected for prophylaxis when the first signs of disease development are observed. Preferably, the diseased retina comprises activated stellate cells to which stem cells are targeted. Therefore, early treatment of the eye is beneficial when there is associated neuroglia. Alternatively, the retina can be treated by administering autologous stem cells after stimulating local proliferation of laser activated astrocytes.

Hematopoietic stem cells are stem cells that can develop into a variety of blood cell types, such as B cells, T cells, granulocytes, platelets, and red blood cells. Lineage surface antigens are a group of cell surface proteins that are markers of mature blood cell lineages. , CD45RA, murine Ly-6G, murine TER-119, CD56, CD64, CD68, CD86 (B7.2), CD66b, human leukocyte antigen DR (HLA-DR) and CD235a (glycoporin A). Hematopoietic stem cells that do not express significant levels of these antigens are generally called lineage negative (Lin ⁻ ). Human hematopoietic stem cells generally express other surface antigens such as CD31, CD34, CD117 (c-kit) and / or CD133. Murine hematopoietic stem cells generally express other surface antigens such as CD34, CD117 (c-kit), Thy-1 and / or Sca-1.

The present invention provides isolated hematopoietic stem cells that do not express significant levels of "lineage surface antigen" (Lin) on the cell surface. These cells present in the "Grid voice" or "Lin ^-" The hematopoietic stem Sephora. In particular, the present invention provides a population of Lin ^- hematopoietic stem cells (Lin ^- HSC), including endothelial progenitor cells (EPC), which can be incorporated into the developing vascular system and then differentiate into vascular endothelial cells. Preferably, this isolated Lin ^- HSC population is present in a culture medium such as phosphate buffered saline (PBS).

As used herein and in the claims, the term "adult" in relation to bone marrow refers to the bone marrow isolated after birth, ie, the bone marrow isolated between adults in infancy and is an expression that contrasts with an embryo. Thus, the term "mammal adult" refers to both childhood mammals and fully mature mammals.

Isolated mammalian lineage negative hematopoietic stem cell (Lin ^- HSC) populations of the invention include endothelial progenitor cells (EPC). The isolated Lin ^- HSC population preferably comprises mammalian cells that express surface antigen CD31, wherein at least about 20% of the cells are normally present in endothelial cells. In another embodiment, at least about 50% of the cells, more preferably at least about 65%, more preferably at least about 75% of the cells express CD31. Preferably, at least about 50% of the cells of the Lin ^- HSC population of the invention express an integrin α6 antigen.

In a preferred embodiment of the rat Lin ^- HSC population, at least about 50% of the cells express a CD31 antigen and at least about 50% of the cells express a CD117 (c-kit) antigen. Preferably, at least about 75%, more preferably at least about 81% of Lin ^- HSC cells express surface antigen CD31. In another preferred embodiment of the rat, at least about 65% of the cells, more preferably about 70% of the cells express surface antigen CD117. A particularly preferred aspect of the invention is a population of rat Lin ^- HSCs in which about 50% to about 85% of the cells express surface antigen CD31 and about 70% to about 75% of the cells express surface antigen CD117.

Another preferred embodiment is the human Lin ^- HSC population in which the cells are CD133 negative, at least about 50% of the cells express the CD31 surface antigen, and at least about 50% of the cells express the integrin α6 antigen. Another preferred embodiment is a human Lin ^- HSC population in which cells are CD133 positive, less than about 30% of the cells express the CD31 surface antigen and less than about 30% of the cells express the integrin α6 antigen.

The isolated Lin ^- HSC populations of the present invention selectively target astrocytes and incorporate into the retinal neovascular system when these cells are injected intravitreally into the eye of an isolated mammalian species, eg, mouse or human.

The isolated Lin ^- HSC population of the present invention includes endothelial progenitor cells that differentiate into endothelial cells to produce vascular structures in the retina. In particular, the Lin ^- HSC population of the present invention is useful for the treatment of retinal neovascular disease and retinal vascular degeneration disease and repair of retinal vascular injury. Lin ^- HSC cells of the invention promote neuronal rescue of the retina and promote upregulation of anti-apoptotic genes. Surprisingly, it has been found that adult Lin ^- HSC cells of the invention can inhibit retinal degeneration even in severe combined immunodeficiency (SCID) mice suffering from retinal degeneration. Normal mouse retinas are predominantly simplified, but almost all cells rescued after treatment with Lin-HSC are cones. In addition, the Lin ^- HSC population can be used to treat retinal defects present in the eyes of newborn mammals, such as mammals suffering from oxygen-induced retinopathy or immature retinopathy.

The invention also isolates a lineage-negative hematopoietic stem cell population comprising endothelial progenitor cells from the mammalian bone marrow and injects the isolated stem cells into the vitreous into the eye of the mammal in sufficient numbers to stop the disease. Including, it provides a method for treating eye diseases of a mammal. The method can be used to treat ocular diseases such as retinal degeneration disease, retinal degenerative disease, ischemic retinopathy, vascular bleeding, vascular leakage and choroidal disease in neonatal, childhood or fully mature mammals. Examples of such diseases include retinal damage as well as age-related macular degeneration (ARMD), diabetic retinopathy (DR), putative ocular histoplasmosis (POHS), immature retinopathy (ROP), sickle cell anemia and retinal pigmentation. Included.

The number of stem cells injected into the eye is sufficient to stop the patient's eye disease state. For example, the number of cells may be effective in restoring eye retinal damage, stabilizing the retinal neovascular system, maturing the retinal neovascular system, and preventing or restoring vascular leakage and vascular bleeding.

Cells in the Lin ^- HSC population of the present invention are therapeutically useful genes, such as genes encoding anti-angiogenic proteins for use in ocular cell-based gene therapy, and neurons that enhance neuronal rescue effects. The gene may be transfected with a gene encoding a nutritional agent.

Transfected cells can include any gene that is therapeutically useful for treating retinal disease. In a preferred embodiment, the transfected Lin ^- HSC of the present invention comprises a TrpRS or an anti-angiogenic fragment thereof as described in detail in co-owned co-pending US Patent Application No. 10 / 080,839, incorporated herein by reference (eg, TrpRS) Genes operably encoding anti-angiogenic peptides, including proteins or protein fragments such as T1 and T2 fragments. Transfected Lin ^- HSCs encoding antiangiogenic peptides of the invention are useful for the treatment of retinal diseases involving abnormal angiogenesis such as diabetic retinopathy and similar diseases. Preferably, Lin ^- HSC is a human cell.

In another preferred embodiment, the transfected Lin ^- HSC of the present invention is a neuronal growth factor, neurotrophin-3, neurotrophin-4, neurotrophin-5, ciliated neurotrophic factor, retinal pigment epithelial derived neurotrophic factor Genes operably encoding neurotrophic agents such as insulin-like growth factors, glial cell derived neurotrophic factors, brain-derived neurotrophic factors, and the like. These neurotrophic Lin ^- HSCs are useful for the promotion of neuronal rescue and retinal nerve damage in retinal neurodegenerative diseases such as glaucoma and retinal pigment degeneration. Transplantation of ciliated neurotrophic factors has been reported to be useful for the treatment of retinal pigment degeneration (Kirby et al., 2001, Mol. Ther. 3 (2): 241-8; Farrar et al., 2002, EMBO Journal 21: 857-864]. Brain-derived neurotrophic factors regulate growth-related genes in injured retinal ganglia. Fournier et al., 1977, J. Neurosci. Res. 47: 561-572. Glioblastoma-derived neurotrophic factors have been reported to retard photoreceptor degeneration in retinal pigment degeneration. McGee et al., 2001, Mol Ther. 4 (6): 622-9].

The present invention also provides a method for isolating lineage negative hematopoietic stem cells including endothelial progenitor cells from mammalian bone marrow. The method comprises the steps of (a) extracting bone marrow from an adult mammal; (b) separating a plurality of monocytes from the bone marrow; (c) monocytes are expressed by one or more lineage surface antigens, preferably CD2, CD3, CD4, CD11, CD11a, Mac-1, CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, Ly-6G (rat ), TER-119 (rat), CD45RA, CD56, CD64, CD68, CD86 (B7.2), CD66b, human leukocyte antigen DR (HLA-DR) and CD235a (glycoporin A) Labeling with biotin-conjugated line panel antibody to the antigen; And (d) a population of lineage negative hematopoietic stem cells that remove monocytes positive for said at least one lineage surface antigen from a plurality of monocytes and that contain endothelial progenitor cells, preferably at least about 20% of the cells express CD31. Recovering the same.

If Lin ^- HSC is isolated from adult bone marrow, monocytes are lineage surface antigens CD2, CD3, CD4, CD11a, Mac-1, CD14, CD16, CD19, CD33, CD38, CD45RA, CD68, CD86 (B7.2) and CD235a It is preferably labeled with a biotin-conjugated lineage panel antibody against. When Lin ^- HSCs are isolated from murine adult bone marrow, monocytes are preferably labeled with biotin-conjugated lineage panel antibodies against lineage surface antigens CD3, CD11, CD45, Ly-6G and TER-119.

In a preferred method, cells are isolated from adult bone marrow and further separated by CD133 lineage. One preferred method of isolating human Lin ^- HSCs further comprises labeling monocytes with biotin-conjugated CD133 antibodies and recovering the CD133 positive Lin ^- HSC population. Typically, less than about 30% of these cells express CD31 and less than about 30% of these cells express integrin α6. The human CD133 positive Lin ^- HSC population of the invention can target a site of peripheral ischemia-induced angiogenesis when injected into an eye without angiogenesis.

Another preferred method of isolating human Lin ^- HSCs further comprises labeling monocytes with biotin-conjugated CD133 antibody, removing CD133 positive cells, and then recovering the CD133 negative Lin ^- HSC population. Typically, at least about 50% of these cells express CD31 and at least about 50% of these cells express integrin α6. The human CD133 negative Lin ^- HSC population of the present invention can be incorporated into the developing vascular system when injected into the eye where angiogenesis is progressing.

The present invention also provides a method of treating ocular angiogenic diseases by administering intraocularly injected into the eye transfected Lin ^- HSC cells of the present invention. Such transfected Lin ^- HSC cells are anti- include ^{HSC-transfected} with useful genes therapeutically Lin, such as a gene encoding angiogenesis or neurotrophic gene product. Preferably, the transfected Lin ^- HSC cells are human cells.

Preferably, at least about 1 × 10 ⁵ Lin ^- HSC cells or transfected Lin ^- HSC cells are administered by intravitreal injection into a mammalian eye suffering from retinal degeneration disease. The number of cells to be injected will depend on the severity of retinal degeneration, the age of the mammal, and other factors that can be readily appreciated by those skilled in the art in the treatment of retinal disease. Lin ^- HSCs may be administered in a single dose, as determined by the treating clinician, or in multiple doses over a period of time.

Lin ^- HSC of the present invention is useful for the treatment of retinal damage and retinal defects involving retinal neuronal degeneration or disruption or degradation of the retinal vascular system. Human Lin ^- HSCs can also be used to produce genetically identical cell lines, ie clones, that are useful for regenerative or repairing retinal vascular systems as well as for the treatment or alleviation of retinal neuronal degeneration.

Example  1. Cell isolation and proliferation; rat Lin ^- HSC  Preparation of Populations A and B

General process . All in vivo assessments were performed in accordance with NIH guidelines for the care and use of laboratory animals, and all assessment processes were conducted by The Scripps Research Institute (TSRI, La Jolla, CA) Animal Care and Use Committee. Approved by the 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).

폴리슈크로스 구배 (제조원: Sigma, St. Louis, MO)를 사용하여 밀도 구배 분리에 의해 분리하고, 마우스의 Lin^- 선별을 위해 바이오틴-접합된 계통 패널 항체 (CD45, CD3, Ly-6G, CD11, TER-119, 제조원: Pharmingen, San Diego, CA)로 표지하였다. 계통 양성 (Lin⁺) 세포를 자기 분리 장치 (AUTOMACS^TM 분리기, 제조원: Miltenyi Biotech, Auburn, CA)를 사용하여 Lin^-HSC로부터 분리하여 제거하였다. 내피 전구세포를 함유하는, 수득된 Lin^-HSC 집단을 다음 항체들을 사용한 FACS^TM Calibur 유세포분석기 (제조원: Becton Dickinson, Franklin Lakes, NJ)를 사용하여 추가로 특성을 규명하였다: PE-접합된-Sca-1, c-kit, KDR 및 CD31 (제조원: Pharmingen, San Diego, CA). Tie-2-GFP 골수 세포를 Tie-2의 특성 규명에 사용하였다.Then monocytes were HISTOPAQUE

Lineage panel antibodies (CD45, CD3, Ly-6G, CD11) isolated by density gradient separation using a polysucrose gradient (Sigma, St. Louis, MO) and biotin-conjugated for Lin ^- selection of mice , TER-119, manufactured by Pharmingen, San Diego, Calif.). Lineage positive (Lin ⁺ ) cells were removed and removed from Lin ^- HSC using a magnetic separation device (AUTOMACS ^™ separator, Miltenyi Biotech, Auburn, Calif.). The resulting Lin ^- HSC population containing endothelial progenitor cells was further characterized using a FACS ^™ Calibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ) using the following antibodies: PE-conjugated-Sca -1, c-kit, KDR and CD31 from Pharmingen, San Diego, CA. Tie-2-GFP bone marrow cells were used to characterize Tie-2.

To harvest adult mouse endothelial cells, mesenteric tissues are surgically removed from ACTbEGFP mice and placed in collagenase (Worthington, Lakewood, NJ) to degrade these tissues and then filtered using a 45 μm filter. It was. The flow was collected and incubated with endothelial growth medium (Clonetics, San Diego, Calif.). Endothelial properties were confirmed by observing cobblestone appearance and staining with CD31 mAb (Pharmingen) and examining the culture for the formation of tubular structures in the MATRIGEL ^™ matrix (Beckton Dickinson, Franklin Lakes, NJ).

Rat Lin ^- HSC Population A. Bone marrow cells were extracted from ACTbEGFP mice by the general procedure described above. Lin ^- 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 Figure 1c. About 81% of Lin ^- HSCs showed CD31 markers, about 70.5% of Lin ^- HSCs showed c-kit markers, about 4% of Lin ^- HSCs showed Sca-1 markers, and about 2.2% of Lin ^- HSC showed a Flk-1 marker and about 0.91% of Lin ^- HSC cells showed a Tie-2 marker. In contrast, Lin ⁺ HSCs isolated from these bone marrow cells had significantly different cellular marker profiles (ie, CD31: 37.4%; c-kit: 20%; Sca-1: 2.8%; Flk-1: 0.05%) )

Rat Lin ^- HSC Population B. Bone marrow cells were extracted from Balb / C, ACTbEGFP and C3H mice by the general procedure described above. Lin ^- HSC cells were characterized by cell surface markers (Sca-1, KDR, c-kit, CD34, CD31 and various integrins: α1, α2, α3, α4, α5, α6, α _M , α _V , α _X , α _IIb , β ₁ , β ₂ , β ₃ , β ₄ , β ₅ and β ₇ ) were analyzed. The results are shown in Table 1 below.

Example  2. Cells in a Rat Model Glass body  administration

Sharp blades were used to create gaps in the mouse eyelids to expose P2 to P6 eyes. Lineage negative HSC population A of the present invention (about 10 ⁵ cells in about 0.5 μl to about 1 μl of cultivation of broth culture medium) was then injected into the vitreous body using a syringe of a 33-gauge (Hamilton, Reno, NV) needle. .

Example  3. EPC  Transfection

배지 (제조원: Invitrogen, Carlsbad, CA) 속에 현탁시켰다. 이어서, DNA (약 1㎍)와 FuGENE 시약(약 3㎕) 혼합물을 첨가하고, 혼합물을 약 18 시간 동안 약 37℃에서 항온처리하였다. 항온처리 후, 세포를 세척하고 수집하였다. 당해 시스템의 형질감염 비율은 약 17%였으며 이는 FACS 분석으로 확인하였다. T2 생산을 웨스턴 블롯팅으로 확인하였다. His₆-태그된 T2-TrpRS의 아미노산 서열은 서열번호 2(도 8)로서 제시되어있다.Rat Lin ^- HSC (Group A) was DNA encoded T2 fragment of TrpRS containing His ₆ tag using FuGENE ^™ 6 transfection reagent (Roche, Indianapolis, IN) according to the manufacturer's protocol (SEQ ID NO: 1). , FIG. 7). Opti-MEM containing Lin ^- HSC cells (approximately 10 ⁶ cells per ml) containing stem cell factor (PeproTech, Rocky Hill, NJ)

Suspended in medium (manufactured by Invitrogen, Carlsbad, Calif.). Then a mixture of DNA (about 1 μg) and FuGENE reagent (about 3 μl) was added and the mixture was incubated at about 37 ° C. for about 18 hours. After incubation, cells were washed and collected. The transfection rate of this system was about 17% and confirmed by FACS analysis. T2 production was confirmed by western blotting. The amino acid sequence of His ₆ -tagged T2-TrpRS is shown as SEQ ID NO: 2 (FIG. 8).

Example  4. Immunohistochemistry and Confocal  analysis

소프트웨어(Bio-Rad)를 사용하여 3차원 영상을 생성시켜, 망막의 온조직 표본 내에서 3가지의 상이한 혈관 발달 층을 검사하였다. 공초점 현미경검사로 구별되는, 증가된 GFP(eGFP) 마우스 및 GFAP/wtGFP 마우스 사이의 GFP 픽셀 강도의 차이를 이용하여 3 차원 영상을 만들었다.Mouse retinas were harvested at various time points and prepared for warm tissue specimen preparation or freeze sectioning. For warm tissue specimens, the retina was fixed in 4% paraformaldehyde and blocked in 50% fetal bovine serum (FBS) and 20% standard goat serum for 1 hour at room temperature. The retina was processed for primary anti-primary and detected with secondary antibody. Primary antibodies used were anti-collagen IV (Chemicon, Temecula, CA), anti-β-gal (Promega, Madison, WI), anti-GFAP (Dako Cytomation, Carpenteria, CA), anti-α-smooth muscle actin ( α-SMA, Dako Cytomation). The secondary antibody used was conjugated to Alexa 488 or 594 fluorescent markers (Molecular Probes, Eugene, OR). Images were captured using an MRC 1024 confocal microscope (Bio-Rad, Hercules, Calif.). LASERSHARP

Three-dimensional images were generated using software (Bio-Rad) to examine three different vascular developmental layers in the warm tissue specimen of the retina. Three-dimensional images were made using differences in GFP pixel intensity between increased GFP (eGFP) mice and GFAP / wtGFP mice, distinguished by confocal microscopy.

Example  5. Mouse In vivo  Retinal Angiogenesis Quantitation Assay

For T2-TrpRS analysis, the primary vascular and deep plexus were reconstructed from three-dimensional images of the mouse retina. The primary vascular plexus was divided into two classes: normal development or stationary vascular progression. The class of inhibition of deep vascular development has been described based on percent vascular inhibition (%), including the following criteria: Complete inhibition of deep plexus formation is marked “complete” and includes normal vascular development (including less than 25% inhibition). ) Are marked as "normal" and the remainder as "partial". For rd / rd mouse rescue data, four individual deep plexus regions were captured using a 10 × lens in each retina of the warm tissue sample. The total length of the vascular system was calculated for each image and summarized and compared between the groups. To obtain accurate information, Lin ^- HSC was injected into one eye and Lin ⁺ HSC was injected into the other eye of the same mouse. Non-injected control retinas were taken from the same mice.

Example  6. Adult Retinal Injury Rat Model

Laser and scar models were made mechanically using diode lasers (150 mW, 1 s, 50 mm) or by puncturing the mouse retina with a 27 gauge needle. Five days after injury, cells were injected by the intravitreal method. After 5 days the eyes were harvested from the mice.

Example  7. Neurotrophic Remedies for Retinal Regeneration

Lineage negative hematopoietic stem cells (Lin ^- HSC) derived from adult rat bone marrow have an angiogenic and neurotrophic rescue effect in a mouse retinal degeneration model. About 0.5 μl containing about 10 ⁵ Lin ^- HSCs of the invention into the vitreous was injected into the vitreous into the right eye of a 10-day-old mouse and evaluated for the presence of the retinal vascular system and nuclear count of the neuronal layer two months later. Nearly the same number of Lin ⁺ HSCs were injected as controls in the left eye of the same mouse and evaluated similarly. As shown in FIG. 9, the retinal vascular system appeared almost normal in Lin ⁻ HSC treated eyes, the inner nuclear layer appeared almost normal, and the outer nuclear layer (ONL) had about 3-4 nuclear layers. In contrast, the contralateral Lin ⁺ HSC treated eye had a markedly shrunken middle retinal vascular layer, a completely shrunk outer retinal vascular layer, the inner nuclear layer was significantly shrunk and the outer nuclear layer completely disappeared. This was dramatically illustrated in mouse 3 and mouse 5. There was no rescue effect in mouse 1, even in about 15% of mice injected.

When visual function was assessed by retinal potential (ERG), repair of positive ERG was observed when both vascular rescue and neuronal rescue were observed (mouse 3 and 5). No positive ERG was observed in the absence of vascular rescue or neuronal rescue (mouse 1). This correlation between vascular and neurotrophic rescue of rd / rd mouse eye by Lin ^- HSC of the present invention is shown in the regression chart shown in FIG. 10. Correlation between nerve recovery (y-axis) and vascular recovery (x-axis) was observed in the medial vascular system type (r = 0.45) and the deep vascular system (r = 0.67).

11 shows that there is no statistically significant correlation between vascular and neuronal rescue by Lin ⁺ HSC. Vascular rescue is quantified and data is shown in FIG. 12. Data for mice 1 month (1M), 2 months (2M) and 6 months (6M) post-injection, shown in FIG. In eyes (black bars) treated with Lin ^- HSC of the present invention, it is demonstrated that blood vessel length was significantly increased, especially at 1 month and 2 months after injection. The neurotrophic rescue effect was quantified by counting nuclei in the inner and outer nuclear layers about two months after the injection of Lin ^- HSC or Lin ⁺ HSC. The results are shown in FIGS. 13 and 14.

Example  8. People Lin ^- HSC  group

폴리슈크로스 구배(Sigma, St. Louis, MO)를 사용하여 밀도구배 분리로 단핵구를 분리했다. 사람 골수 단핵세포로부터 Lin^-HSC 집단을 분리하기 위해, 자기 분리 시스템 (AUTOMACS™ 분류기, 제조원: Miltenyi Biotech, Auburn, CA)하에 다음과 같은 바이오틴-접합된 계통 패널 항체를 사용했다: CD2, CD3, CD4, CD11a, Mac-1, CD14, CD16, CD19, CD33, CD38, CD45RA, CD64, CD68, CD86, CD235a(Pharmingen).Bone marrow cells were extracted from healthy adult volunteers following the general procedure described above. Then, HISTOPAQUE

Monocytes were isolated by density gradient separation using a polysucrose gradient (Sigma, St. Louis, Mo.). To isolate the Lin ^- HSC population from human bone marrow monocytes, the following biotin-conjugated line panel antibodies were used under a magnetic separation system (AUTOMACS ™ sorter, Miltenyi Biotech, Auburn, Calif.): CD2, CD3, CD4, CD11a, Mac-1, CD14, CD16, CD19, CD33, CD38, CD45RA, CD64, CD68, CD86, CD235a from Pharmingen.

Based on CD133 expression, the human Lin ^- HSC population was further classified into two subgroups. Cells were labeled with biotin-conjugated CD133 antibodies and sorted into CD133 positive and CD133 negative subpopulations.

Example  9. Resection of Human and Rat Cells in a Rat Retinal Degeneration Model Glass body  administration

C3H / HeJ, C3SnSmn.CB17-Prkdc SCID and rd10 mouse species were used as retinal degeneration models. C3H / HeJ and C3SnSmn.CB17-Prkdc SCID mice (The Jackson Laboratory, Maine) are homozygous for retinal degeneration 1 (rd1) mutation, a mutation that causes early onset severe retinal degeneration. This mutation is located in exon 7 of the Pde6b gene encoding the simplified photoreceptor cGMP phosphodiesterase β subunit. Mutations in this gene have been found in human patients with autosomal recessive retinal pigment degeneration (RP). In addition, C3SnSmn.CB17-Prkdc SCID mice were homozygous for the severe complex immunodeficiency spontaneous mutation (Prkdc SCID) and were used in human cell delivery experiments. Retinal degeneration in rd10 mice is caused by mutations in exon 13 of the Pde6b gene. It is also a clinically relevant RP model that develops later than rd1 / rd1 and has mild retinal degeneration. All assessments were performed in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals, and all procedures were conducted by The Scripps Research Institute (TSRI, La Jolla, Calif.) Animal Care and Use Committee. Was approved by

Sharp blades were used to create gaps in the mouse eyelids to expose P2 to P6 eyes. Subsequently, lineage negative HSC cells (about 10 ⁵ cells in about 0.5 μl to about 1 μl of cell culture medium) of rat population A or human population C were transferred using a syringe of 33-gauge (Hamilton, Reno, NV) needle. The eye was injected intravitreally. To visually identify injected human cells, cells were labeled with dye (cell tracer green CMFDA, Molecular Probes) prior to injection.

소프트웨어(Bio-Rad)를 사용하여 포착했다. 혈관계 정량을 위해, 4개의 독립적 영역 (900㎛ x 900㎛)을 중간 혈관층 또는 심부 혈관층의 중앙부로부터 무작위로 선택하고, LASERPIX

분석 소프트웨어(Biorad)를 사용하여 혈관계의 총 길이를 측정했다. 동일한 혈관총에서의 이러한 4 영역의 총 길이는 이후 분석에 이용했다.Retinas were harvested at various time points and fixed with 4% paraformaldehyde (PFA) and methanol and then blocked for 1 hour at room temperature in 50% FBS / 20% NGS. To stain the retinal vascular system, the retina was incubated with anti-CD31 (Pharmingen) and anti-collagen IV (Chemicon) antibodies, followed by Alexa 488 or 594 conjugated secondary antibodies (Molecular Probes, Eugene, Oregon). The retina was planarized with 4 radial relaxation ablation for warm tissue specimen preparation. Imaging of the vascular system of the medial or deep retinal plexus [Dorrell et al., 2002 Invest Ophthalmol. Vis. Sci. 43: 3500-3510] Radiance MP2100 Confocal Microscope and LASERSHARP

Captured using software (Bio-Rad). For vascular quantitation, four independent regions (900 μm × 900 μm) were randomly selected from the middle of the middle vascular layer or the deep vascular layer and LASERPIX

Analysis software (Biorad) was used to measure the total length of the vascular system. The total length of these four regions in the same vascular plexus was later used for analysis.

Flat sampled retinas were re-embedded to make frozen sections. The retina was left overnight in 4% PFA and then incubated with 20% sucrose. This retina was embedded in an optimal cleavage temperature compound (OCT: Tissue-Tek; Sakura FineTech, Torrance, CA). Frozen sections (10 μm) were rehydrated in PBS containing nuclear dye DAPI (Sigma-Aldrich, St. Louis, Missouri). DAPI labeled nuclear images of the other three regions (280 μm wide, fair sampling) of a single section containing the optic nerve papilla and the entire peripheral retina were captured by confocal microscopy. The number of nuclei located in the ONL of three independent regions within one section was counted and aggregated for analysis. Simple linear regression analysis was performed to investigate the relationship between the vascular system length of the deep plexus and the number of nuclei in the ONL.

After overnight dark acclimatization, mice were anesthetized by intraperitoneal injection of ketamine 15 μg / gm and xylazine 7 μg / gm. The retinal potential (ERG) was recorded from the corneal surface of each eye after pupil dilation (1% atropine sulfate) using gold loop corneal electrodes along with oral reference and tail ground electrodes. The stimulation was caused by a Grass light stimulator (PS33 Plus, Grass Instruments, Quincy, Mass.) Mounted on the highly reflective Ganzfeld dome. Simplified responses to short wavelength (Wratten 47A; λ _max = 470 nm) flashes were recorded at intensities in the range below the maximum that could be imparted by the optical stimulator (0.668 cd-s / m 2). The reaction signal was amplified (CP511 AC amplifier, Grass Instruments), quantified (PCI-1200, National Instruments, Austin, TX) and analyzed by computer. For each mouse, ERGs from treated and untreated eyes were recorded and used as their internal control. 100 measurements were averaged for the weakest signal. The mean response of untreated eyes was subtracted numerically from the response of treated eyes, and the difference in these signals was taken as an index of functional rescue.

소프트웨어(SiliconGenetics, Redwood City, CA)를 사용하여 유전자 발현을 분석했다. 정제된 사람 또는 마우스 HSC를 P6 마우스의 유리체내로 주사했다. P45에서, 망막을 절제하고, 1) 사람 HSC-주사된 구제된 마우스 망막, 2) 사람 HSC-주사된, 구제되지 않은 마우스 망막, 및 3) 마우스 HSC-주사된, 구제된 마우스 망막의 분획으로 나누어 수집하여, RNA 정제와 사람-특이적 U133A 아피메트릭스 칩 하이브리드화에 사용했다. GENESPRING

소프트웨어를 사용하여 배경값 이상으로 발현되고 사람 HSC 구제된 망막에서 더 많이 발현되는 유전자를 동정했다. 이러한 각 유전자에 대해 프로브-쌍 발현 프로필을 독립적으로 분석하고, dChip을 이용한 정상인의 U133A 마이크로어레이 실험 모델과 비교하여, 사람 종 특이적인 하이브리드화를 측정하고, 교차 종(cross-species) 하이브리드화에 의한 거짓 양성을 배제했다.Microarray analysis was used to assess the expression of Lin ^- HSC targeted retinal genes. P6 rd / rd mice were injected with Lin ⁻ or CD31-HSC. The retinas of these mice were excised in RNase free medium 40 days after injection (rescue of the retinal vascular system and photoreceptor layer is apparent at this point after injection). One-quarter of each retina was analyzed with a warm tissue sample to confirm normal HSC targeting as well as vascular and neuronal protection. Successfully injected retina RNA was purified using TRIzol (Life Technologies, Rockville, MD), phenol / chloroform RNA isolation protocol. RNA was hybridized to Affymetrix Mu74Av2 chip and GENESPRING

Gene expression was analyzed using software (SiliconGenetics, Redwood City, CA). Purified human or mouse HSCs were injected into the vitreous of P6 mice. At P45, the retina is excised and divided into fractions of 1) human HSC-injected rescued mouse retina, 2) human HSC-injected, unsaved mouse retina, and 3) mouse HSC-injected, rescued mouse retina The aliquots were collected and used for RNA purification and human-specific U133A Affymetrix chip hybridization. GENESPRING

The software was used to identify genes that are expressed above background and more expressed in human HSC rescued retinas. For each of these genes, we independently analyze the probe-pair expression profile, measure human species-specific hybridization, and compare cross-species hybridization with the dChip's U133A microarray experimental model. Ruled out false positives.

21 shows flow cytometry data comparing the expression of CD31 and integrin alpha 6 surface antigens in the CD133 positive (DC133 ⁺ ) and CD133 negative (CD133 ⁻ ) human Lin ^- HSC populations of the present invention. The left panel shows flow cytometry scatter plots. Middle and right panels are histograms showing specific antibody expression levels for a cell population. The Y axis represents the number of events and the X axis represents the strength of the signal. The schematic histogram is an isotype IgG control antibody showing non-specific background staining levels. The filled histogram shows the level of specific antibody expression for the cell population. Shifting the filled histogram to the right of the coarse (control) histogram indicates an increase in antibody expression and fluorescent signal above the background level. Comparing the position of the peaks of the filled histogram between the two cell populations, there is a difference in protein expression in that cell. For example, CD31 is expressed above background levels in both CD133 ⁺ and CD133 ⁻ cells of the invention; More cells express low levels of CD31 in the CD133 ⁺ cell population than in the CD133 ⁻ population. From these data, CD31 expression varies between the two populations, and alpha 6 integrin expression is mainly limited to cells in the Lin ⁻ population, so it can be used as a marker of cells with vascular- and neurotrophic rescue functions.

Intravitreal injection of CD133 positive and CD133 negative Lin ^- HSC subpopulations into the eyes of neonatal SCID mice showed the highest levels of incorporation into the developing vascular system in CD133 negative subpopulations expressing both CD31 and integrin α6 surface antigens. 21, see bottom). CD133 positive subpopulations (FIG. 21, top) that do not express CD31 or integrin α6 target the site of peripheral ischemia-induced angiogenesis, but not when injected into the eye where the angiogenesis progresses.

The rescued and non-rescued retinas were immunohistochemically analyzed using antibodies specific for either Simplified or Conical Opsin. The same eye used for the ERG recording shown in FIG. 17 was analyzed for either Simplified or Conical Obsin. In the wild-type mouse retina, less than 5% of the photoreceptors present are cones (Soucy et al . 1998, Neuron 21: 481493), using red / green cone opsin as shown in FIG. 25 (A) or FIG. 25 (B The immunohistochemical staining observed using Simplified Rhodopsin as shown in Fig. 6) was consistent with the above percentage of vertebral cells. Antibodies specific for Simplified Rhodopsin (rho4D2) were provided by Dr. Robert Molday of the University of British Columbia and used as previously described (Hicks et al. 1986, Exp. Eye Res. 42: 5571). Rabbit antibodies specific for the vertebral red / green opsin were purchased from the manufacturer (Chemicon (AB5405)) and used according to the manufacturer's instructions.

Example  10. Rat Cells in a Rat Model for Oxygen-Induced Retinal Degeneration Glass body  administration

As an oxygen-induced retinal degeneration (OIR) model, newly born wild type CD57B16 mice were exposed to hyperoxia (75% oxygen) between P7 and P12 days after birth. 22 shows normal postnatal vascular development of C57B16 mice from P0 to P30. At P0 only superficial vessels germinating around the optic disc were observed. Over the next few days, the primary superficial network was extended to the periphery, reaching far away in the P10. Between P7 and P12, a secondary (deep) plexus developed. P17 had extensive superficial and deep vascular networks (FIG. 22, inset). Thereafter, remodeling occurred with the development of the tertiary (intermediate) vascular layer until approximately adult structure at P21.

In contrast, in the OIR model, the normal developmental sequence of events was severely disrupted after 75% oxygen exposure to P7 to P12 (FIG. 23). At P3, the adult rat Lin ^- HSC population of the invention was injected intravitreally into one eye of the mouse, and then the eye of this mouse was treated with OIR and the other eye was injected with PBS or CD31 negative cells as a control. . FIG. 24 shows that the Lin ^- HSC population of the present invention can reverse the degeneration effect of high oxygen levels in the developing mouse retina. Fully developed superficial and deep retinal vascular systems were observed in P17 in the treated eye, whereas control eyes showed a wide avascular region with virtually no deep blood vessels (FIG. 24). About 100 mouse eyes were observed in the OIR model. Normal angiogenesis was observed in 58% of eyes treated with the Lin ^- HSC population of the present invention, whereas 3% in control eyes treated with PBS and 12% in control eyes treated with CD31 ⁻ cells showed normal angiogenesis.

Results and discussion

Vascular development of the rat retina; Ocular Angiogenesis Model

Mouse eyes provide a recognized model for the study of vascular development in mammalian retinas, eg, vascular development in human retinas. During development of the rat retinal vascular system, ischemia-induced retinal vessels develop in close association with astrocytic cells. These glial elements travel radially from the optic disc to the ganglion cell layer and onto the retina of the third fetal human fetus or rodent newborn. As the rat retinal vessels develop, endothelial cells use this already established astrocytic template to determine the vascular pattern of the retina (see FIGS. 1A and 1B). 1 (a and b) show schematic views of the developing mouse retina. FIG. 1A shows the development of a primary aneurysm (dark line at the top left of the figure) superimposed on an astrocytic template (blurred line), and FIG. 1B shows the second phase of retinal angiogenesis. In FIG. 1, GCL means ganglion cell layer, IPL means inner vascular plexus layer, INL means inner nuclear layer; OPL means outer plexus layer; ONL means outer nuclear layer; RPE means retinal pigment epithelium; ON means optic nerve; P means the periphery.

At birth, the retinal vascular system is virtually nonexistent. By 14 days of age (P14), the retina develops with visual acuity and at the same time develops complex primary (superficial) and secondary (deep) layers of retinal vessels. First, the spoke-like peripheral blood vessels grow radially toward the periphery beyond the existing stellate cell network, gradually connecting to each other to form a capillary plexus. These blood vessels grow as monolayers in nerve fibers through P10 (FIG. 1A). A side branch extending from P7 to P8 extends out of this primary plexus and penetrates through the retina into the outer plexus layer, where it forms a secondary or deep retinal plexus. The entire network up to P21 is extensively remodeled and a tertiary or intermediate vasculature is formed at the inner surface of the inner nuclear layer (FIG. 1B).

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, the presence of giant astrocytic templates before the appearance of endogenous blood vessels allows one to assess the role for cell-cell targeting during neovascularization. In addition, this consistent and reproducible vascular process of neonatal retina is known to be induced by hypoxia, and in this respect has similarities to many retinal diseases known to be involved in ischemia.

Bone marrow-derived endothelial progenitor cells ( EPC )of Enrichment

Although cell surface marker expression has been evaluated in detail for the EPC populations found in HSC preparations, the markers uniquely identifying EPC are still very few defined. To enrich for EPCs, hematopoietic system marker positive cells (Lin ⁺ ), namely B lymphocytes (CD45), T lymphocytes (CD3), granulocytes (Ly-6G), monocytes (CD11) and red blood cells (TER-119) Of bone marrow mononuclear cells. The Sca-1 antigen was used to further enrich EPC. When comparing the results obtained after intravitreal injection of the same number of Lin ^- HSC Sca-1 ⁺ or Lin ^- cells, no difference was detected between the two groups. Indeed, when only Lin ^- Sca-1 ^- cells were injected, much greater incorporation into the developing blood vessels was observed.

Based on the functional assay, the Lin ^- HSC population of the present invention is rich in EPC. In addition, the Lin ⁺ HSC population behaves quite differently from the Lin ^- HSC population in terms of functionality. For each fraction (based on already reported in vitro characterization studies) epitopes commonly used to identify EPCs were also evaluated. None of these markers were exclusively associated with the Lin ⁻ fraction, but all increased about 70 to about 1800% in Lin ⁻ HSC compared to the Lin ⁺ HSC fraction (FIG. 1C). 1C shows the flow cytometry characteristics of Lin ⁺ HSC and Lin ^- HSC isolated cells derived from bone marrow. The top row of FIG. 1C shows the hematopoietic stem cell dot plot distribution of non-antibody labeled cells. R1 represents the quantifiable-gated area of positive PE-staining; R2 represents GFP-positive. The dot plot of Lin ^- HSC is shown in the middle row and the dot plot of Lin ⁺ HSC is shown in the bottom row. C57B / 6 cells were labeled with PE-conjugated antibodies against Sca-1, c-kit, Flk-1 / KDR, CD31. Tie-2 data was obtained from Tie-2-GFP mice. The percentage of the corner of the dot plot represents the percentage of positively-labeled cells in the entire Lin ⁻ or Lin ⁺ HSC population. Interestingly, adopted EPC markers such as Flk-1 / KDR, Tie-2 and Sca-1 were poorly expressed and therefore could not be used for further fractionation.

Intravitreal Injected HSC Lin ^- cells contain EPCs that target astrocytes and are incorporated into the vascular system of the developing retina . Lin ^- HSC injected into the vitreous isolate from the bone marrow of adult (GFP or LacZ transgenic) mice to target specific cell types in the retina, determine whether they can use astrocytic templates and be involved in retinal angiogenesis About 10 ⁵ cells or Lin ⁺ HSC (control, about 10 ⁵ cells) from the Lin ^- HSC composition of the present invention were injected into mouse eyes 2 days after birth (P2). At day 4 post-injection (P6), a number of cells from the Lin ^- HSC compositions of the invention derived from GFP or LacZ transgenic mice were attached to the retina and had a characteristic elongated shape of endothelial cells (FIG. 2A). 2 shows engraftment of Lin ⁻ cells into the developing mouse retina. As shown in FIG. 2A, eGFP + Lin ^- HSC injected intravitreally at day 4 post-injection (P6) attaches to and differentiates on the retina.

In many areas of the retina, GFP-expressing cells were arranged in a pattern similar to basal stellate cells and in blood vessels. These fluorescent cells were observed in front of the endogenous developing vascular network (FIG. 2B). In contrast, only a few Lin ⁺ HSCs (FIG. 2C) or adult mouse mesenteric endothelial cells (FIG. 2D) were attached to the retinal surface. In order to determine if cells from the injected Lin ^- HSC composition can also attach to the retina with already established blood vessels, we injected the Lin ^- HSC composition into adult eyes. Interestingly, no cells were attached to the retina or incorporated into established normal retinal vessels (FIG. 2E). This indicates that the Lin ^- HSC composition of the present invention does not disrupt the normally developed vascular system and does not initiate abnormal angiogenesis in the normally developed retina.

To determine the relationship between the injected Lin ^- HSC composition of the present invention and retinal astrocytes, transgenic mice expressing glial fibrous acidic proteins (GFAP, markers of astrocytes) and promoter-induced green fluorescent protein (GFP) Was used. Retinal testing of these GFAP-GFP transgenic mice injected with Lin ^- HSCs from eGFP transgenic mice demonstrated co-localization of the injected eGFP EPC with conventional astrocytic cells (FIGS. 2F-2H, arrows). . The process of eGFP + Lin ^- HSC was observed to match the basal astrocytic network (arrow, FIG. 2G). These eye tests demonstrated that only the labeled cells injected were attached to astrocytes, and in the P6 mouse retina where the periphery of the retina does not yet have endogenous blood vessels, the injected cells are astrocytic cells in these regions that do not yet form blood vessels. Was observed attached to. Surprisingly, the labeled cells injected were observed in the depths of the retina and at the exact location (normal arrows) in which normal retinal vessels would later develop.

In order to determine if the injected Lin ^- HSC of the present invention is stably incorporated into the developing retinal vascular system, retinal vessels were then examined at various time points. As early as P9 (7 days after injection), Lin ⁻ HSC was incorporated into the CD31 ⁺ structure (FIG. 2J). By P16 (14 days after injection), cells have already been extensively incorporated into the vascular-like structure of the retina (FIG. 2K). If Rhodamine-dextran was injected into the blood vessels (to identify functional retinal vessels) prior to sacrificing the animal, most Lin ^- HSCs were aligned with the parental vessels (FIG. 2L). The following two patterns of labeled cell distribution were observed: (1) in one pattern the cells were scattered along the blood vessels between the unlabeled endothelial cells, and (2) the other pattern consisted only of cells with all the blood vessels labeled. It was lost. The injected cells were also incorporated into the blood vessels of the deep vascular plexus (FIG. 2M). Sporadic incorporation of Lin ^- HSC-derived EPCs into the neovascular system has already been reported, but it is the first report that the vascular network consists entirely of these cells. This demonstrates that cells of the bone marrow-derived Lin ^- HSC population of the present invention injected into the vitreous can be efficiently incorporated into all layers of the retinal plexus that is being formed.

Histological examination of non-retinal tissues (eg brain, liver, heart, lung, bone marrow) did not demonstrate the presence of any GFP positive cells when tested up to 5 or 10 days after intravitreal injection. . This suggests that a subset of cells in the Lin ^- HSC fraction can be stably incorporated into the developing retinal vascular system by selectively targeting retinal stellate cells. Because these cells have many features of endothelial cells (associated with retinal stellate cells, elongated shape, stable incorporation into the parent vessel and not present in extravascular locations), these cells are EPCs present in the Lin ^- HSC population. Indicates. Targeted astrocytic cells are the same type as observed in many hypoxic retinopathies. It is well known that glial cells are the major component of neovascular fronds observed in DR and other types of retinal damage. Under conditions of reactive gliosis and ischemia-induced angiogenesis, activated astrocytes proliferate and produce cytokines to produce GFAP, similar to that observed during neonatal retinal angiogenesis in many mammalian species, including humans. Adjust up.

Since the Lin ^- HSC composition of the present invention will target activated astrocytes in adult mouse eyes as well as in neonatal mouse eyes, Lin ^- HSC cells may be photocoagulated (FIG. 3A) or the tip of the needle (FIG. 3B) damaged retina. Injected into the eye. In both models, markedly GFAP stained cell populations were observed only around the site of injury (FIGS. 3A and 3B). Cells from the injected Lin ^- HSC composition were concentrated at the site of injury and remained specifically associated with GFAP-positive astrocytes (FIGS. 3A and 3B). At these sites, Lin ^- HSC cells were also observed to migrate into the depths of the retina to levels similar to those observed during deep retinal vasculogenesis in newborns. The intact portion of the retina did not contain Lin ^- HSC cells as observed when injecting Lin ^- HSC into normal intact adult retina (FIG. 2E). These data show that the Lin ^- HSC composition can selectively target activated glial cells in the neonatal retina as well as in the injured adult retina showing neuroglia.

Lin ^- HSC injected into the vitreous can rescue and stabilize the degenerative vascular system. Since the Lin ^- HSC composition injected into the vitreous targets the astrocytes and is incorporated into the normal retinal vascular system, these cells also stabilize the degenerative vascular system in ischemic or retinal degeneration diseases associated with neuroglia and vascular degeneration. rd / rd mice are a model for retinal degeneration showing severe degeneration of the photoreceptor and retinal vascular layers up to 1 month after birth. The retinal vasculature of these mice develops normally up to P16, at which point the deep vascular plexus degenerates, and in most mice the deep and intermediate plexus nearly degenerated to P30.

To determine if HSCs can rescue degenerative blood vessels, Lin ⁺ HSC or Lin ^- HSC (from Balb / c mice) was injected intravitreally into rd / rd mice at P6. Until P33, after injection of Lin ⁺ cells, almost all of the deepest vessels of the retina were absent (FIGS. 4A and 4B). In contrast, most retinas injected with Lin ^- HSC had a nearly normal retinal vascular system with three comparable, well-formed vascular layers up to P33 (FIGS. 4A and 4D). Quantification of this effect demonstrated that the average length of blood vessels in the deep plexus of rd / rd eyes injected with Lin ⁻ was nearly three times longer than the eyes injected with untreated or Lin ⁺ cells (FIG. 4E). Surprisingly, injection of Lin ^- HSC composition derived from rd / rd adult mouse (FVB / N) bone marrow also rescued the degenerative rd / rd neonatal mouse retinal vascular system (FIG. 4F). Degeneration of the vascular system in the rd / rd mouse eye was observed as early as 2 to 3 weeks after birth. Injection of Lin ^- HSC at a point as late as P15 partially stabilized the degenerative vascular system in rd / rd mice for more than 1 month (FIGS. 4G and 4H).

Lin ^- HSC compositions injected into younger (eg P2) rd / rd mice were also incorporated into the developing superficial vasculature. By P11, these cells migrated to the level of the deep plexus and formed the same pattern as the pattern observed in the wild type outer retinal vascular layer (FIG. 5A). To more clearly describe how cells from the injected Lin ^- HSC composition were incorporated into and stabilized in the retinal vascular system in rd / rd mice, the Lin ^- HSC composition derived from Balb / c mice was Tie-2-GFP. FVB mouse eyes were injected. FVB mice have an rd / rd genotype and all endogenous blood vessels fluoresce because they express the fusion protein Tie-2-GFP.

If unlabeled cells from the Lin ^- HSC composition were injected into the neonatal Tie-2-GFP FVB eye and subsequently incorporated into the developing vascular system, endogenous Tie-2-GFP labeled blood vessels were injected and incorporated into the labeled unlabeled cells. There should be an unlabeled gap corresponding to Lin ^- HSC. Subsequent staining with other vascular markers (eg, CD-31) then outlines the entire vessel, thus determining whether the non-endogenous endothelial vessel is part of the vascular system. Two months after injection, CD-31 positive and Tie-2-GFP negative blood vessels were observed in the retina of the eye injected with Lin ^- HSC composition (FIG. 5B). Interestingly, most of the rescued vessels contained Tie-2-GFP positive cells (FIG. 5C). The distribution of perivascular cells, as measured by smooth muscle actin staining, was not changed by Lin ^- HSC injection, regardless of whether there was vascular rescue (FIG. 5D). These data clearly demonstrate that the Lin ^- HSC compositions of the invention injected intravitreally migrate into the retina of genetically deficient mice and participate in the formation of normal retinal blood vessels to stabilize the endogenous degenerative vascular system.

Inhibition of retinal angiogenesis by cells transfected from Lin ^- HSC . Most retinal vascular diseases involve abnormal vascular proliferation rather than vascular degeneration. Transgenic cells targeted to astrocytes can be used to deliver anti-angiogenic proteins and inhibit angiogenesis. Cells from the Lin ^- HSC composition were transfected with T2-tryptophanyl-tRNA synthetase (T2-TrpRS). T2-TrpRS is a 43kD fragment of TrpRS that strongly inhibits angiogenesis of the retina (FIG. 6A). At P12, the retina of the eye injected with the control plasmid-transfected Lin ^- HSC composition (not containing the T2-TrpRS gene) at P2 had normal primary (FIG. 6C) and secondary (FIG. 6D) retinal vascular guns. . When evaluated 10 days after injection of T2-TrpRS transfected Lin ^- HSC composition of the present invention into P2 eye, the primary network had significant abnormal characteristics (FIG. 6E) and the formation of the deep retinal vascular system was almost completely inhibited ( 6f). The few vessels observed in these eyes were markedly weakened due to the large gaps between them. The degree of inhibition by Lin ^- HSC cells secreting T2-TrpRS is described in detail in Table 2 below.

T2-TrpRS is produced and secreted by the cells in the Lin ^- HSC composition in vitro, and after injection of these transfected cells into the vitreous, a 30kD fragment of T2-TrpRS was observed in the retina (FIG. 6B). Such 30kD fragments are especially observed only in the retinas injected with the transfected Lin ^- HSC of the present invention, and this apparent molecular weight reduction compared to recombinant or in vitro-synthesized proteins is due to processing or degradation of T2-TrpRS in vivo. It may be. These data indicate that the Lin ^- HSC composition can be used to deliver functionally active genes, such as genes expressing angiostatic molecules, to the astrocytic cells to target the retinal vascular system. Although the observed angiogenesis effect is likely due to cell-mediated activity, this is unlikely to occur because the eye treated with the Lin ^- HSC composition that was identical but not transfected with T2 had a normal retinal vascular system.

Vascular Inhibition by Lin ^- HSC Secreting T2-TrpRS Primary vascular gun Deep veins Suppressed Normal Full partial normal TsTrpRS
(15 eyes) 60% 40%
(9 eyes) (6 eyes) 33.3% 60% 6.7%
(5 eyes) (9 eyes) (1 eye) Control group
(13 eyes) 0% 100%
(0 eyes) (13 eyes) 0% 38.5% 61.5%
(0 eyes) (5 eyes) (8 eyes)

Lin ^- HSC populations injected intravitreally concentrated into retinal astrocytes and incorporated into the blood vessels and may be useful in the treatment of many retinal diseases. Most of the cells from the injected HSC composition attach to the astrocytic template, but a few migrate deep into the retina, subsequently entering areas where deep vascular networks will develop. Although no GFAP-positive astrocytic cells were observed in this region 42 days before birth, this does not exclude the possibility that GFAP-negative glial cells are already present and can provide a signal for Lin ^- 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.

Since cells from the injected Lin ^- HSC composition specifically adhered to GFAP-expressing glial cells, the Lin ^- HSC composition of the present invention can be used to target preangiogenic lesions in the retina, regardless of the type of injury. . For example, in ischemic retinopathy such as diabetes, angiogenesis is a response to hypoxia. By targeting the Lin ^- HSC composition to a pathological site of angiogenesis, the developing neovascular system can be stabilized to prevent neovascular abnormalities, such as bleeding or edema (cause of DR-related vision loss), and initially prevent angiogenesis. It can potentially reduce irritated hypoxia. Abnormal blood vessels may return to their normal state. In addition, vascular inhibitory proteins such as T2-TrpRS can be delivered to the pathological angiogenesis site using the transfected Lin ^- HSC composition and laser-induced activation of astrocytes. Since laser photocoagulation is commonly used in clinical ophthalmology, this approach is applied for many retinal diseases. This cell-based approach has been studied in cancer therapy, and it is more advantageous to use it for eye diseases because intraocular injections can deliver numerous cells directly to the disease site.

Neurotrophic and Angiotrophic Remedies by Lin ^- HSC . MACS was used to isolate Lin ^- HSCs from the bone marrow of increased green fluorescent protein (eGFP), C3H (rd / rd), FVB (rd / rd) mice as described above. Lin ^- HSCs containing EPCs from these mice were injected intravitreally into P6 C3H or FVB mouse eyes. Retinas were collected at various time points (1 month, 2 months and 6 months) after injection. Vascular systems were examined by confocal laser scanning microscopy after staining with antibody to CD31 and retinal histology after nuclear staining with DAPI. In addition, microarray gene expression analysis of mRNA from the retina at various time points was used to identify genes potentially involved in this effect.

The eyes of rd / rd mice had severe degeneration of both the sensory nerve retina and the retinal vascular system up to P21. The eyes of rd / rd mice treated with Lin ^- HSC at P6 maintained normal retinal vascular system for 6 months, and both the deep and middle layers were markedly at all time points (1 month, 2 months and 6 months) compared to the control group. Improved (see FIG. 12). In addition, the inventors found that the retina treated with Lin ^- HSC was thicker than the eye treated with Lin ⁺ HSC as a control (1 month: 1.2 times, 2 months: 1.3 times, 6 months; 1.4 times) externally. It was observed that the nuclear layer had a greater number of cells (1 month: 2.2 times, 2 months: 3.7 times, 6 months: 5.7 times). "Rescue" (eg Lin ^- HSC) large-scale genomic analysis compared to control (untreated or treated with non-Lin ^- HSC) rd / rd retina, correlated with sHSP (small open heat protein), and vascular and neuronal rescue Significant upregulation of genes encoding specific growth factors with, eg, the genes encoding the proteins described in panels A and B of FIG. 20 was demonstrated.

The bone marrow-derived Lin ^- HSC population of the present invention induced remarkably and reproducible maintenance of normal vascular system in rd / rd mice and dramatically increased photoreceptors and other neuronal layers. This neurotrophic rescue effect correlates with significant upregulation of small thermal proteins and growth factors, thus providing insight into the therapeutic approach of untreated retinal degenerative diseases at present.

rd1 / rd1 mouse retinas show severe vascular and neuronal degeneration. The development of blood vessels and neurons in the normal postnatal retina of mice has been fully explained and is similar to the changes observed in the human fetus at the third and third month. Dorrell et al., 2002 Invest Ophthalmol. Vis. Sci. 43: 3500-3510. Mice homozygous for the rd1 gene share many features that appear in human retinal degeneration (Frassson et al., 1999, Nat. Med. 5: 1183-1187], a rapid photoreceptor (PR) loss with severe vascular atrophy as a result of mutation of the gene encoding PR cGMP phosphodiesterase. Bowes et al. 1990, Nature 347: 677-680. To investigate the vascular system during retinal development and subsequent degeneration, antibodies to collagen IV (CIV), extracellular matrix (ECM) proteins of mature vascular system, and CD31 (PECAM-1), markers of endothelial cells, were used (FIG. 15). The retina of rd1 / rd1 (C3H / HeJ) developed normally until about 8 days after birth (P8), when the degeneration of the photoreceptor-containing outer nuclear layer (ONL) begins. This ONL denatured rapidly, resulting in cell death due to apoptosis, leaving only one nuclear layer at P20. Warm tissue sampled retinas were double-stained with antibodies to both CIV and CD31, followed by Blanks et al., 1986, J. Comp. Neurol. 254: 543-553, in which vascular degeneration in rd1 / rd1 mice similar to that described in detail was confirmed. The primary and deep retinal vascular layers appeared to develop normally by P12, but then there was a sharp loss of endothelial cells as evidenced by the absence of CD31 staining. CD31 positive endothelial cells existed in a normal distribution until P12 but then disappeared rapidly. Interestingly, CIV positive staining remained present for the time point investigated, suggesting that blood vessels and related ECMs formed normally, but only substrate remained after P13 where no CD31 positive cells were observed (FIG. 15, middle panel). . In addition, the medial plexus degenerated after P21, but its progression was slower than that observed in the deep plexus (FIG. 15, top panel). Retinal vascular and neuronal layers of normal mice were presented together compared to rd1 / rd1 mice (right panel, FIG. 15).

Neuroprotective Effect of Bone Marrow-derived Lin ^- HSC in rd1 / rd1 Mice . Intravitreal injected Lin ^- HSC is incorporated into the endogenous retinal vascular system in a total of three vascular plexuses to prevent degeneration of the blood vessels. Interestingly, the injected cells were virtually not observed in the outer nuclear layer. These cells were observed to be incorporated into or adjacent to the forming retinal vessels. Rat Lin ^- HSC (derived from C3H / HeJ) was injected intravitrealally into P3 immediately before the onset of degeneration in C3H / HeJ (rd1 / rd1) mouse eyes. By P30, control cell (CD31 ⁻ ) -injected eyes exhibited a typical rd1 / rd1 phenotype, ie almost complete degeneration of the deep plexus and ONL was observed in all retinas examined. Eyes injected with Lin ^- HSC maintained a moderate and deep plexus that appeared normal. Surprisingly, significantly more cells were observed in the internuclear layer (INL) and ONL of Lin ^- HSC-injected eye than in control cell-injected eye (FIG. 16A). This salvage effect of Lin ^- HSC was observed at 2 months (FIG. 16B) and up to 6 months after injection (FIG. 16C). Differences in the neuronal-containing INL and ONL as well as the vasculature of the medial and deep plexus of Lin ^- HSC injected eye were remarkable at all time points measured when compared to rescued eye and unrelieved eye (FIG. 16B and FIG. 16C). This effect was quantified by measuring the total length of the vascular system (FIG. 16D) and counting the number of DAPI positive cell nuclei observed in ONL (FIG. 16E). Simple linear regression analysis was applied to the data at all time points.

In Lin ^- HSC injected eyes, a statistically significant correlation was observed between vascular rescue and neuronal (eg ONL thickness) rescue at P30 (p <0.024) and P60 (p <0.034) (FIG. 16F). This correlation was maintained at a high level (P <0.14), although there was no statistical significance (p <0.14) at P180 when comparing the Lin ^- HSC-injected retina with the control-cell injected retina. In contrast, there was no significant correlation between the preservation of the vascular system and the preservation of ONL at any time in the control cell-injected retina (FIG. 16F). These data demonstrate that intravitreal injection of Lin ^- HSC simultaneously results in retinal vascular and neuronal rescue in the retina of rd1 / rd1 mice. The injected cells were not observed at any location or ONL other than in or near the retinal vessel.

Lin ^- Functional Rescue of HSC Injected rd / rd Retina. Retinal potentials (ERG) were performed on mice 2 months old after injection of control cells or rat Lin ^- HSC (FIG. 17). Immunohistochemistry and microscopic analyzes were performed on each eye after ERG recordings to confirm that vascular and neuronal rescue occurred. Representative ERG recordings obtained from treated and untreated eyes of control group showed that the signal (treated eyes-untreated eyes) with the numerically subtracted signal from the rescued eye was apparent with an amplitude of about 8 to 10 microvolts. It showed that it produces a signal that can be detected easily (Fig. 17). Obviously, the signals from both eyes are seriously abnormal. However, constant and detectable ERG was recorded from Lin ^- HSC treated eyes. In all cases ERG could not be detected from control eyes. The amplitude of the signal recorded in the rescued eye was significantly less than normal, but this signal was consistently observed with histological relief and was similar to the magnitude of the amplitude reported in other gene-based rescue studies. Taken together, these results demonstrate that some functional relief occurred in the eye treated with Lin ^- HSC of the present invention.

The rescued rd / rd retinal cell types are mainly cones . The rescued and non-rescued retinas were immunohistochemically analyzed using antibodies specific for either Simplified or Conical Opsin. The same eye used for the ERG recording shown in FIG. 17 was analyzed for either Simplified or Conical Obsin. In the wild-type mouse retina, less than 5% of the photoreceptors present are cones (Soucy et al . 1998, Neuron 21: 481-493), using red / green cone opsin as shown in FIG. 25 A or in FIG. As shown, the immunohistochemical staining pattern observed using Simplified Rhodopsin was consistent with the above percentage of vertebral cells. When wild-type retinas were stained with pre-immunogen IgG, no staining was observed anywhere in the sensory nerve retina other than autofluorescence of blood vessels (FIG. 25C). At 2 months postnatally, the retinas of non-injected rd / rd mice were substantially atrophic, showing no staining with antibodies to red / green cone opsin (FIG. 25 D) or rhodopsin (FIG. 25 G). Had an outer nuclear layer. Eyes injected with CD31-HSC, a control, were also not stained positive for the presence of vertebrae (FIG. 25 E) or hepatic (FIG. 25 H) opsin. In contrast, the contralateral eye injected with Lin-HSC had about 3 to about 8 rows of nuclei in the preserved outer nuclear layer; Most of these cells were positive for vertebral opsin (FIG. 25 F), and about 1 to 3% were positive for hepatic opsin (FIG. 25 I). Specifically, this is almost the opposite of what is commonly observed in normal mouse retinas predominantly simplified. These data demonstrate that injection of Lin-HSC preserves the vertebrae for long periods of time where the vertebrae can normally denature.

Lin ^- HSC from human bone marrow ( hBM ) also rescues retinal degeneration. Lin ^- HSC isolated from human bone marrow acts similarly to rat Lin ^- HSC. Bone marrow was collected from human donors and Lin ⁺ HSC removed to obtain a human Lin ^- HSC (hLin ^- HSC) population. These cells were labeled ex vivo with fluorescent dyes and injected into C3SnSmn.CB17-Prkdc SCID mouse eyes. The hLin ^- HSC injected in this way moved to and targeted the retinal angiogenesis site in the same manner as observed when injected with mouse Lin ^- HSC (FIG. 18A). In addition to vascular targeting, human Lin ^- HSC also provided potent rescue effects on both the vascular and neuronal layers of rd1 / rd1 mice (FIGS. 18B and 18C). This observation confirms the presence of cells in the human bone marrow that can target the retinal vascular system and prevent retinal degeneration.

Lin ^- HSC shows vascular and neurotrophic effects in rd10 / rd10 mice . rd1 / rd1 mice are the most widely used and best characterized models of retinal degeneration (Chang et al. 2002, Vision Res. 42: 517-525, but degeneration is so fast that this differs from the usual slow progression observed in human disease. In this line, photoreceptor cell degeneration is initiated around P8, a time when the retinal vascular system is still rapidly growing (FIG. 15). Subsequent degeneration of the deep retinal vascular system occurs even during the formation of an intermediate plexus, so that the retinas of rd1 / rd1 mice never develop completely, as observed in most people with this disease. The rd10 mouse model was used to study Lin ^- HSC mediated vascular rescue, because the progress of degeneration is slow and more similar to human retinal degeneration. In rd10 mice, photoreceptor cell degeneration begins around P21 and vascular degeneration begins immediately thereafter.

Since normal neurosensory retinal development is mostly completed in P21, degeneration is observed to begin after retina completes differentiation, in this regard it is more similar to human retinal degeneration than the rd1 / rd1 mouse model. Lin ^- HSC or control cells from rd10 mice were injected into P6 eyes and the retina was evaluated at various time points. Eye-derived retinas injected with Lin ^- HSC and control cells at P21 appeared to be normal with all vascular layers fully developing and INL and ONL developing normally (FIGS. 18D and 18H). Retinal degeneration at about P21 was initiated and progressed with aging. At P30, the control cell injected retina showed severe vascular and neuronal degeneration (FIG. 18I) and the Lin ^- HSC injected retina retained nearly normal vascular layer and photoreceptor cells (FIG. 18E). The difference between the rescued and non-saved eyes was more pronounced at later observations (compare FIG. 18 (F and G) with FIG. 18 (J and K)). Progression of vascular degeneration in control treated eyes was very clearly observed by immunohistochemical staining for CD31 and collagen IV (FIGS. 18I-18K). The control treated eye was almost completely negative for CD31, while the collagen IV positive blood vessel "track" remained clearly, suggesting that vascular degeneration occurred rather than incomplete blood vessel formation. In contrast, Lin ^- HSC treated eyes had both CD31 and collagen IV positive blood vessels, which looked very similar to normal wild type eyes (compare FIGS. 18F and 18I).

Gene expression analysis of rd / rd mouse retina after Lin ^- HSC treatment. Rescue and non-rescue retinas were analyzed using large scale genomics (microarray analysis) to identify putative mediators of neurotrophic rescue. Gene expression of rd1 / rd1 mouse retinas treated with Lin ^- HSC was compared to gene expression of the non-injected retina and control cells (CD31 ⁻ ) injected retina. Each of these comparisons was repeated three times. To be considered present, the expression level of the gene had to be at least twice higher than the background level in a total of three replicates. Genes three-fold upregulated in Lin ⁻ HSC-protected retinas compared to control cell-injected rd / rd mouse retinas and non-injected rd / rd mouse retinas are shown in panels A and B of FIG. 20. Coefficient of variation (COV) levels were calculated for expressed genes by dividing the standard deviation by the average expression level of each cRNA copy. In addition, the correlation between expression levels and noise fluctuations was calculated by correlating the mean and standard deviation (SD) to each other. Correlation between gene expression levels and standard deviations for each gene has been obtained, which allows to determine background and reliable expression level thresholds. In total, the data naturally fall within acceptable ranges (Tu et al . 2002, Proc. Natl . Acad . Sci. USA 99: 14031-14036). The genes described below each exhibited expression levels above these critical expression levels. Paired "t-test" values for the described genes are shown in Table 1. In each case, the p-value is reasonable (nearly less than 0.05), which demonstrates similarities between replicas and that there may be significant differences between different test groups. Most of the significantly upregulated genes, including MAD and Ying Yang-1 (YY-1) (Austen et al. 1997, Curr. Top. Microbiol . Immunol . 224: 123-130), protect cells from apoptosis. Encode a protein with a function associated with it. A number of crystallin genes with similar function and sequence homology to known heat-like proteins involved in protecting cells from stress were also upregulated by Lin ^- HSC treatment. Expression of α-crystallin was shown to be limited to ONL by immunohistochemical analysis (FIG. 19). 19 shows that crystallin αA is upregulated in rescued outer nuclear layer cells after treatment with Lin ⁻ HSC but not in contralateral eyes treated with control cells. Left panel shows IgG staining (control) in rescued retina. The middle panel shows crystallin αA in the rescued retina. The right panel shows crystallin αA in the non-saved retina.

Messenger RNAs from rd1 / rd1 mouse retinas rescued with human Lin ^- HSCs were hybridized to human specific Affymetrix U133A microarray chips. After rigorous analysis, a large number of genes whose mRNA expression is human specific, higher than background levels, and significantly higher in human Lin ^- HSC rescued retinas compared to rat Lin ^- HSC rescued retinas and human control cell injected non- rescued retinas Was confirmed (FIG. 20, Panel C). This evaluation was made by this microarray bioinformatics technique, since both CD6, a cell adhesion molecule expressed on the surface of early and newly differentiated CD34 + hematopoietic stem cells, and another gene expressed by hematopoietic stem cells, were revealed by this microarray bioinformatics technique. The validity of the protocol was confirmed. In addition, several growth factors and neurotrophic factors were also expressed above background levels in human Lin ^- HSC rescued mouse retinal samples (FIG. 20, Panel D).

Markers for lineage-related hematopoietic cells were used to negatively select a population of Lin ^- HSCs from bone marrow containing EPCs. Although a subset of bone marrow-derived Lin ^- HSCs that can be used as EPCs is not characterized by commonly used cell surface markers, the action of these cells in developing or damaged retinal vascular systems is dependent on Lin ⁺ or adult endothelial cell populations. It is entirely different from the action observed. These cells are selectively targeted to retinal angiogenesis sites to participate in the formation of parent blood vessels.

Hereditary retinal degeneration disease is often accompanied by a loss of the retinal vascular system. Effective treatment of such a disease requires not only functional recovery but also maintenance of complex tissue structures. Several recent studies have documented the use of cell-based delivery of trophic factors or the stem cells themselves, some combination of both may be necessary. For example, the use of growth factor therapy to treat retinal degeneration disorders results in uncontrolled overgrowth of blood vessels resulting in severe disruption of normal retinal tissue structure. The use of neuronal or retinal stem cells to treat retinal degenerative diseases can reconstruct neuronal function, but a functional vascular system may be necessary to maintain the functional integrity of the retina. Incorporation of cells from the Lin ^- HSC composition of the present invention into the retinal vessels of rd / rd mice stabilized the denatured vascular system without disrupting the retinal structure. This rescue effect was also observed when the cells were administered to P15 rd / rd mice. Since vascular degeneration is initiated at P16 in rd / rd mice, this observation expands the therapeutic range for effective Lin ^- HSC treatment. Retinal neurons and photoreceptors are preserved and visual function is maintained in the eye injected with Lin ^- HSC of the present invention.

Adult bone marrow-derived Lin ^- HSCs exert significant vascular and neurotrophic effects upon intravitreal injection into mice with retinal degeneration. This remedy lasts up to 6 months after treatment and is most effective when Lin ^- HSC is injected before retinal degeneration is complete (usually up to 30 days postnatally up to 16 days postnatally in mice showing complete retinal degeneration). This rescue is observed in a retinal degeneration model of two mice, surprisingly achieved by adult bone marrow derived HSCs when the recipient is an immunodeficient rodent with retinal degeneration (eg, SCID mice) or when the donor is a mouse with retinal degeneration. Can be. Some recent reports have described partial phenotypic rescues following virus-based gene rescue with wild-type genes in mice or dogs with retinal degeneration [Al, 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 provides the first general cell-based therapeutic effect achieved by vascular rescue. Thus, the potential utility of such an attempt to treat a group of diseases (eg, retinal pigment degeneration) in which there are more than 100 known related mutations is much more practical than the individual gene therapies developed to treat each known mutation.

The exact molecular mechanism of the neurotrophic rescue effect is unknown, but this is only observed if there is simultaneous vascular stabilization / rescue. That is, the presence of the injected stem cells itself is not sufficient to cause neurotrophic rescue, and the apparent absence of stem cells derived from stem cells in the outer nuclear layer eliminates the possibility of transforming the injected cells with photoreceptors. Data obtained by microarray gene expression analysis demonstrated significant upregulation of genes known to have anti-apoptotic effects. Since most neuronal cell death observed in retinal degeneration is due to apoptosis, such protection may be a significant therapeutic advantage in prolonging the lifespan of photoreceptors and other neurons critical for visual function in these diseases. c-myc is a transcription factor involved in apoptosis by upregulation of various downstream apoptosis inducing factors. c-myc expression was increased 4.5-fold in rd / rd mice compared to wild-type, suggesting a potential association with photoreceptor degeneration observed in rd1 / rd1 mice. Two rapidly upregulated genes Mad1 and YY-1 in Lin ^- HSC protected retinas (FIG. 20, Panel A) are known to inhibit the activity of c-myc and thus inhibit c-myc induced apoptosis. do. In addition, overexpression of Mad1 was found to inhibit Fas-induced activation of caspase-8, another major component of the apoptosis pathway. Thus, upregulation of these two molecules may play an important role in protecting the retina from vascular and neuronal degeneration by preventing the onset of apoptosis, which generally leads to degeneration in rd / rd mice.

Another set of genes that are significantly upregulated in Lin ^- HSC protected retinas include members of the crystallin family (FIG. 20, Panel B). Similar to fissure proteins and other stress induced proteins, crystallins can be activated by retinal stress and provide a protective effect against apoptosis. Abnormally low expression of αA-crystallin correlates with loss of photoreceptors in rat retinal dystrophy models, and recent proteomic analysis of rd / rd mouse retinas shows crystallinity in response to retinal degeneration. Demonstrated upregulation of. Based on our microarray data of EPC rescued rd / rd mouse retinas, upregulation of crystallins appears to play a major role in neuroprotection of EPC mediated retinas.

Genes such as c-myc, Mad1, Yx-1, and crystallin will be downstream mediators of neuronal rescue. Although microarray analysis of retinas rescued with mouse stem cells herein did not demonstrate increased levels of known neurotrophic factors, neurotrophic agents can modulate anti-apoptotic gene expression. On the other hand, analysis of stem cell mediated rescue from human bone marrow using human specific chips demonstrates a low but significant increase in multiple growth factor gene expression.

Upregulated genes include several members of the fibroblast growth factor family and otoferlin. Mutations in autoperlin genes are associated with genetic diseases that induce hearing loss due to auditory neuropathy. Autoperlin production by injected Lin ^- HSC may also contribute to the prevention of retinal neuropathy. Historically, vascular changes observed in patients with retinal degeneration and animals have long been estimated to be concomitant with reduced metabolic requirements following photoreceptor death. The data of the present invention suggest that at least in mice with hereditary retinal degeneration, preservation of the normal vascular system may help maintain components of the outer nuclear layer. The concept supported in the recent literature report is that tissue-specific vasculature exhibits nutritional effects beyond what is expected by simply providing vascular "nutrition". For example, hepatic endothelial cells may be induced after VEGFR1 activation to produce growth factors important for hepatic cell regeneration and maintenance despite liver damage (LeCouter et al., 2003, Science 299: 890-893).

Similar interactions between vascular endothelial cells and adjacent liver parenchymal cells have been reported to be involved in liver organogenesis long before the formation of functional blood vessels. The endogenous retinal vascular system of individuals with retinal degeneration cannot very dramatically promote rescue, but if such vascular system is supported by endothelial precursors from the myeloid hematopoietic stem cell population, they can make the vascular system more resistant to degeneration. At the same time, it can promote the survival of vascular cells as well as retinal nerve cells. In people with retinal degeneration, delaying the onset of complete retinal degeneration will provide additional years of vision retention. Animals treated with Lin ^- HSC of the present invention significantly preserved ERG, which may be sufficient for vision support.

Clinically, functional vision is still preserved, but it is well known that photoreceptors and other neurons may be substantially lost. At some point, clinical limits are crossed and vision is lost. Almost everyone's hereditary retinal degeneration develops slowly initially, so identify individuals with retinal degeneration and treat them intravitreally with the autologous bone marrow stem cells of the present invention, thereby delaying retinal degeneration and subsequent vision loss. Can be. In order to increase targeting and incorporation of stem cells of the present invention, the presence of activated astrocytes will be preferred (Otani et al . 2002, Nat. Med . 8: 1004-1010); This can be achieved by using early treatment in the case of neuroglia, or by using a laser to stimulate local proliferation of activated astrocytes.

Optionally, prior to intraocular injection, stem cells may be transfected ex vivo with one or more neurotrophic agents to increase the effect of rescue. This approach can be applied to the treatment of other visual neuronal degenerative diseases such as glaucoma with retinal ganglion cell degeneration.

The Lin ^- HSC population of the present invention contains an EPC population that can promote angiogenesis by targeting reactive astrocytes and can be incorporated into an established template without disrupting the retinal structure. Lin ^- HSC of the present invention also provides a surprising long-term neurotrophic rescue effect in the eye with retinal degeneration. In addition, genetically modified autologous Lin ^- HSC compositions containing EPCs can be implanted into ischemic or abnormally vascularized eyes and stably incorporated into neovascularized blood vessels to continuously deliver therapeutic molecules locally for long periods of time. have. This local delivery of genes that express pharmacological agents at physiologically meaningful doses represents a new paradigm for treating eye diseases that are currently incurable.

For example, the photoreceptors in normal mouse retinas are predominantly simplified, but the outer nuclear layer observed after rescue using Lin-HSC of the present invention predominantly contained vertebral bodies. Most hereditary human retinal degeneration occurs as a result of primary hepatic-specific defects, and loss of the vertebrae is considered secondary to hepatic dysfunction, which is probably related to the loss of any trophic factor expressed by the hepatic. Will be there. The present method of inducing vertebral survival despite vertebral / retinal degeneration promoted by Lin-HSC may provide a better way to preserve vertebral-dominant human macular in diseases such as retinal pigment degeneration.

Numerous variations and modifications of the aspects described above can be made without departing from the spirit and scope of the novel features of the invention. Accordingly, no limitations with respect to the specific aspects illustrated herein should be intended or inferred.

<110> The Scripps Research Institute <120> Isolated lineage negative hematopoietic stem cells and       methods of treatment therewith    <130> TSRI-988.2 PCT <150> US 10 / 933,634 <151> 2004-09-03 <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 4742 <212> DNA <213> Artificial Sequence <220> <223> DNA encoding His-tagged human T2-TrpRS <400> 1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540 tccgctcatg agacaataac 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ggccttttgc tggccttttg ctcacatgtt 2280 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060 gccagcagca tcctgcgatg cagatccgga 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caaccaagtg aaaggcattt tcggcttcac 3960 tgacagcgac tgcattggga agatcagttt tcctgccatc caggctgctc cctccttcag 4020 caactcattc ccacagatct tccgagacag gacggatatc cagtgcctta tcccatgtgc 4080 cattgaccag gatccttact ttagaatgac aagggacgtc gcccccagga tcggctatcc 4140 taaaccagcc ctgttgcact ccaccttctt cccagccctg cagggcgccc agaccaaaat 4200 gagtgccagc gacccaaact cctccatctt cctcaccgac acggccaagc agatcaaaac 4260 caaggtcaat aagcatgcgt tttctggagg gagagacacc atcgaggagc acaggcagtt 4320 tgggggcaac tgtgatgtgg acgtgtcttt catgtacctg accttcttcc tcgaggacga 4380 cgacaagctc gagcagatca ggaaggatta caccagcgga gccatgctca ccggtgagct 4440 caagaaggca ctcatagagg ttctgcagcc cttgatcgca gagcaccagg cccggcgcaa 4500 ggaggtcacg gatgagatag tgaaagagtt catgactccc cggaagctgt ccttcgactt 4560 tcagaagctt gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa 4620 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc 4680 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg 4740 at 4742 <210> 2 <211> 392 <212> PRT <213> Artificial Sequence <220> <223> His-tagged human T2-TrpRS <400> 2 Met Ser Ala Lys Gly Ile Asp Tyr Asp Lys Leu Ile Val Arg Phe Gly  1 5 10 15 Ser Ser Lys Ile Asp Lys Glu Leu Ile Asn Arg Ile Glu Arg Ala Thr             20 25 30 Gly Gln Arg Pro His His Phe Leu Arg Arg Gly Ile Phe Phe Ser His         35 40 45 Arg Asp Met Asn Gln Val Leu Asp Ala Tyr Glu Asn Lys Lys Pro Phe     50 55 60 Tyr Leu Tyr Thr Gly Arg Gly Pro Ser Ser Glu Ala Met His Val Gly 65 70 75 80 His Leu Ile Pro Phe Ile Phe Thr Lys Trp Leu Gln Asp Val Phe Asn                 85 90 95 Val Pro Leu Val Ile Gln Met Thr Asp Asp Glu Lys Tyr Leu Trp Lys             100 105 110 Asp Leu Thr Leu Asp Gln Ala Tyr Gly Asp Ala Val Glu Asn Ala Lys         115 120 125 Asp Ile Ile Ala Cys Gly Phe Asp Ile Asn Lys Thr Phe Ile Phe Ser     130 135 140 Asp Leu Asp Tyr Met Gly Met Ser Ser Gly Phe Tyr Lys Asn Val Val 145 150 155 160 Lys Ile Gln Lys His Val Thr Phe Asn Gln Val Lys Gly Ile Phe Gly                 165 170 175 Phe Thr Asp Ser Asp Cys Ile Gly Lys Ile Ser Phe Pro Ala Ile Gln             180 185 190 Ala Ala Pro Ser Phe Ser Asn Ser Phe Pro Gln Ile Phe Arg Asp Arg         195 200 205 Thr Asp Ile Gln Cys Leu Ile Pro Cys Ala Ile Asp Gln Asp Pro Tyr     210 215 220 Phe Arg Met Thr Arg Asp Val Ala Pro Arg Ile Gly Tyr Pro Lys Pro 225 230 235 240 Ala Leu Leu His Ser Thr Phe Phe Pro Ala Leu Gln Gly Ala Gln Thr                 245 250 255 Lys Met Ser Ala Ser Asp Pro Asn Ser Ser Ile Phe Leu Thr Asp Thr             260 265 270 Ala Lys Gln Ile Lys Thr Lys Val Asn Lys His Ala Phe Ser Gly Gly         275 280 285 Arg Asp Thr Ile Glu Glu His Arg Gln Phe Gly Gly Asn Cys Asp Val     290 295 300 Asp Val Ser Phe Met Tyr Leu Thr Phe Phe Leu Glu Asp Asp Asp Lys 305 310 315 320 Leu Glu Gln Ile Arg Lys Asp Tyr Thr Ser Gly Ala Met Leu Thr Gly                 325 330 335 Glu Leu Lys Lys Ala Leu Ile Glu Val Leu Gln Pro Leu Ile Ala Glu             340 345 350 His Gln Ala Arg Arg Lys Glu Val Thr Asp Glu Ile Val Lys Glu Phe         355 360 365 Met Thr Pro Arg Lys Leu Ser Phe Asp Phe Gln Lys Leu Ala Ala Ala     370 375 380 Leu Glu His His His His His His 385 390

Claims (52)

A pharmaceutical composition comprising a lineage negative hematopoietic stem cell population comprising an isolated, hematopoietic stem cell and an endothelial progenitor cell derived from human bone marrow, wherein the cells of the isolated lineage negative hematopoietic stem cell population are CD133 negative and at least 50% of cells Is expressing a surface antigen to integrin α6 and at least 50% of the cells express surface antigen CD31, the pharmaceutical composition delays cortical cell degeneration of the retina, thereby ischemic retinopathy, angiogenesis in the eye where angiogenesis progresses. Pharmaceutical composition for treating retinal disease selected from the group consisting of bleeding, vascular leakage, choroidal disease, age-related macular degeneration, diabetic retinopathy, putative eye histoplasmosis, immature retinopathy, sickle cell anemia, and retinal pigmentosa . delete delete delete delete delete delete delete delete delete delete delete A pharmaceutical composition comprising a lineage-negative hematopoietic stem cell population comprising an isolated, hematopoietic stem cell and endothelial progenitor cell derived from human bone marrow, wherein the cells of the isolated lineage negative hematopoietic stem cell population are CD133 positive and less than 30% Cells express surface antigens against integrin α6 and less than 30% of the cells express surface antigen CD31, and the pharmaceutical composition delays ischemia-induced peripheral angiogenesis (neovascularization). Angiogenesis is an ischemic retinopathy, vascular hemorrhage, vascular leakage, choroidal disease, age-related macular degeneration, diabetic retinopathy, presumptive ocular histoplasmosis, immature retinopathy, sickle cell anemia and retinal disease in the developing eye. A pharmaceutical composition for treating a retinal disease selected from the group consisting of pigmented degeneration. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete The population of isolated lineage negative hematopoietic stem cells according to claim 1 is characterized in that the population of tryptophanyl-tRNA synthetase T2 fragment (T2-TrpRS), nerve growth factor, neurotropin-3, neurotropin-4, neurotro A peptide or protein selected from the group consisting of pin-5, ciliated neurotrophic factor, neurotrophic factor derived from retinal pigment epithelium, insulin-like growth factor, neurotrophic factor derived from glial cell line and brain-derived neurotrophic factor A pharmaceutical composition that is transfected to produce. delete delete delete delete delete delete delete delete delete delete delete delete delete delete The method of claim 13, wherein the lineage negative hematopoietic stem cell population is (a) separating monocytes from human bone marrow; (b) labeling said monocytes with biotin-conjugated lineage panel antibodies against CD2, CD3, CD4, CD11a, Mac-1, CD14, CD16, CD19, CD33, CD38, CD45RA, CD64, CD68, CD86 and CD235a ; (c) removing monocytes positive for said lineage surface antigen from the monocytes isolated in step (a) and recovering a population of lineage negative hematopoietic stem cells containing endothelial progenitor cells; (d) labeling the monocytes with biotin-conjugated CD133 antibody; And (e) a pharmaceutical composition produced by the method comprising recovering a CD133 positive lineage negative hematopoietic stem cell population. The method of claim 1, wherein the lineage negative hematopoietic stem cell population is (a) separating monocytes from human bone marrow; (b) labeling said monocytes with biotin-conjugated lineage panel antibodies against CD2, CD3, CD4, CD11a, Mac-1, CD14, CD16, CD19, CD33, CD38, CD45RA, CD64, CD68, CD86 and CD235a ; (c) removing monocytes positive for said lineage surface antigen from the monocytes isolated in step (a) and recovering a population of lineage negative hematopoietic stem cells containing endothelial progenitor cells; (d) labeling the monocytes with biotin-conjugated CD133 antibody; (e) removing the CD133 positive lineage negative hematopoietic stem cell population; And (f) a pharmaceutical composition produced by the method comprising recovering a CD133 negative lineage negative hematopoietic stem cell population. The population of isolated lineage negative hematopoietic stem cells according to claim 13 is characterized in that the T2 fragment of tryptophanyl-tRNA synthetase (T2-TrpRS), nerve growth factor, neurotropin-3, neurotropin-4, neurotro A peptide or protein selected from the group consisting of pin-5, ciliated neurotrophic factor, neurotrophic factor derived from retinal pigment epithelium, insulin-like growth factor, neurotrophic factor derived from glial cell line and brain-derived neurotrophic factor A pharmaceutical composition that is transfected to produce.

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