MX2007002639A - Isolated lineage negative hematopoietic stem cells and methods of treatment therewith. - Google Patents

Abstract

Isolated, mammalian, adult bone marrow-derived, lineage negative hematopoietic stem cell population (Lin HSCs) contain endothelial progenitor cells (ECPs) capable of rescuing 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 treatment of ocular vascular diseases and to ameliorate cone cell degeneration in the retina. In a preferred embodiment, the Lin HSCs are isolated by extracting bone marrow from an adult mammal; separating a plurality of monocytes from the bone marrow; labeling the monocytes with biotin-conjugated lineage panel antibodies to one or more lineage surface antigens; removing of monocytes that are positive for the lineage surface antigens from the plurality of monocytes, and recovering a Lin HSCs population containing EPCs. The isolated Lin HSCs also can be transfected with therapeutically useful genes. The tre atment may be enhanced by stimulating proliferation of activated astrocytes in the retina using a laser.

Description

HEMATOPQYETIC STEM CELLS OF ISOLATED NEGATIVE LINEAGE AND METHODS OF TREATMENT WITH THE SAME FIELD OF THE INVENTION The present invention relates to isolated, mammalian stem cells. More particularly, the invention relates to populations of hematopoietic stem cells negative (Lin-HSC) derived from the bone marrow and methods to preserve cone cells in a retina of a mammal suffering from a degenerative ocular disease by treating the eye of the mammal with Lin populations "HSC isolates." BACKGROUND OF THE INVENTION Age-related macular degeneration (ARMD) and diabetic retinopathy (DR) are the main causes of visual loss in industrialized nations and are the result of abnormal retinal neovascularization. Because the retina consists of well-defined layers of neuronal, glial, and vascular elements, relatively small alterations such as those seen in vascular proliferation or edema can lead to a significant loss of visual function.Hereditic retinal degenerations such as Retinitis pigmentosa (RP), are also associated with an vascular ormalities, such as arteriolar narrowing and atrophy vascular. Most hereditary degenerations of the human retina specifically affect rod photoreceptors, but there is also a concomitant loss of cones, the main cellular component of the macula, the region of the retina in humans that is responsible for fine visual acuity, central. The specific survival factors of the cones have been described recently (Mohand-Said et al., 1998, Proc.Nat.Acid.Sci.USA, 95: 8357-8362) and can facilitate the survival of cones in mouse models of retinal degeneration. Hereditary degenerations of the retina affect as many as 1 in 3,500 individuals who are characterized by progressive night blindness, visual field loss, optic nerve atrophy, arteriolar attenuation, altered vascular permeability, and central vision loss that often develop to blindness total (Heckenlively, JR, editor, 1988; Retinitis Pigmentosa, Philadelphia: JB Lippincott Co.). Molecular genetic analysis of these diseases has identified mutations in more than 110 different genes counting for only a relatively small percentage of known affected individuals (Humphries et al., 1992, Science 256: 804-808; Farrar et al., 2002; EMBO J. 21: 857-864). Many of these mutations are associated with enzymatic and structural components of the machinery of phototransduction including rhodopsin, cGMP phosphodiesterase, rds periphery, and RPE65. Despite these observations, there are still no effective treatments to reduce or reverse the evolution of these retinal degenerative diseases. Recent advances in gene therapy have led to the successful reversal of rds phenotypes (Ali et al 2000, Nat. Genet 25: 306-310) and rd (Takahashi et al., 1999, J. Virol. 73: 7812-7816) in mice and the RPE65 phenotype in dogs (Acland et al., 2001, Nat. Genet, 28: 92-95) when the wild type transgene is delivered to photoreceptors or to the pigmented epithelium of the retina (RPE) in animals with a specific mutation . For many years it has been known that a population of stem cells exists in the circulation and bone marrow of a normal adult. Different sub-populations of these cells can be differentiated along positive (Lin +) or negative (Lin ~) hematopoietic lineages. In addition, it has been shown that the population of hematopoietic stem cells (HSC) of negative lineage contains endothelial progenitor cells (EPC) capable of forming blood vessels in vi tro and in vivo (See Asahara et al., 1997, Science 275: 964-7 ). These cells can participate in normal and pathological postnatal angiogenesis (See Lyden et al 2001 Nat. Med. 7, 1194-201; Kalka et al., 2000, Proc. Nati, Acad. Sci. USA 97: 3422-7; and Kocher et al., 2001 Nat. Med. 7: 430-6) as well as differentiate into a variety of non-endothelial cell types including hepatocytes (See Lagasse et al., 2000,? At Med. 6: 1229-34), microglia (See Priller et al. 2002 Na t. Med. 7: 1356-61), cardiomyocytes (See Orlic et al., 2001, Proc. Nati, Acad. Sci. USA 98: 10344-9) and epithelium. { See Lyden et al 2001, Nat. Med. 7: 1194-1201). Although these cells have been used in several experimental models of angiogenesis, the mechanism of CLD that is directed to the neovasculature is not known and no strategy has been identified that effectively increases the number of cells that contribute to a particular vasculature. The hematopoietic stem cells of the bone marrow are currently the only type of stem cells commonly used for therapeutic applications. Bone marrow HSCs have been used in transplants for more than 40 years. Currently, advanced methods for harvesting purified stem cells are under investigation to develop therapies for the treatment of leukemia, lymphoma and hereditary blood disorders. Critical applications of stem cells in humans have been investigated for the treatment of diabetes and advanced kidney cancer in a limited number of patients. SUMMARY OF THE INVENTION The present invention provides a method for alleviate cellular degeneration of cones in the retina of a mammal suffering from an eye disease. The method comprises the step of administering to the mammalian retina a population of isolated negative lineage hematopoietic stem cells derived from mammalian bone marrow, which comprises hematopoietic stem cells and endothelial progenitor cells. The cells are administered in an amount sufficient to delay cellular degeneration of cones in the retina. A preferred method comprises isolating from the bone marrow of a mammal suffering from an ocular disease a population of negative lineage hematopoietic stem cells including endothelial progenitor cells and subsequently intravitreally injecting isolated stem cells into a mammalian eye in a number enough to alleviate the degeneration of cone cells in the retina. The methods of the present invention utilize a population of hematopoietic stem cells of negative lineage (Lin "HSC), mammalian, isolated (ie, hematopoietic stem cells (HSCs) that do not express surface lineage antigens (Lin) on their cell surface) derived of mammalian bone marrow Preferably, the cells are autologous stem cells (ie, derived from the bone marrow of the mammalian subject to be treated).

The population of Lin "isolated, mammalian HSCs includes endothelial progenitor cells (EPCs), also known as endothelial precursor cells, which selectively target the activated astrocytes of the retina when injected intravitreally into the eye. In a preferred embodiment, the Lin "HSC populations of the present invention are isolated by the steps of extracting bone marrow from a mammal suffering from an ocular disease; separating a plurality of monocytes from the bone marrow; label monocytes with a panel of lineage antibodies conjugated with biotin to one or more lineage surface antigens, remove monocytes that are positive for lineage surface antigens and subsequently recover a Lin population "HSC containing EPCs." Preferably the monocytes are labeled with a panel of lineage antibodies conjugated with biotin to one or more lineage surface antigens selected from the group consisting of CD2, CD3, CD4, CD11, CDlla, Mac-1, CD14, CD16, CD19, CD24, CD33, CD36 , CD38, CD45, Ly-6G, TER-119, CD45RA, CD56, CD64, CD68, CD86, CD66b, HLA-DR, and CD235a (Glycoforin A) .Preferably, at least about 20% of the cells in the population Lin "HSC isolated from the present invention expresses the CD31 surface antigen. The isolated cells are then administered to the diseased eye of the mammal, preferably by intraocular injection. In a preferred embodiment, at least about 50% of the Lin "isolated HSCs express the CD31 surface antigen and at least about 50% of the Lin" isolated HSCs express the CD117 surface antigen (c-kit). The EPCs within the Lin population "HSCs of the present invention are extensively incorporated into the developing retinal blood vessels and into the retina neuronal network, and remain stably incorporated into the neovasculature and neural network of the eye. Mouse is predominantly rods, however, in mice treated with the methods of the present invention, cells recovered after treatment with Lin "HSCs were almost all surprisingly cones. In a preferred embodiment, the cells of the isolated "HSC" Lin populations are transfected with a therapeutically useful gene, eg, the cells can be transfected with polynucleotides that functionally code for neurotrophic agents or antiangiogenic agents that selectively target or recognize the neovasculature and inhibit the formation of new vessels without affecting the already established vessels through a form of cell-based gene therapy In one embodiment, the Lin populations "HSC, isolated, useful in the methods of present invention includes a gene encoding a peptide that inhibits angiogenesis. The Lin "HSCs that inhibit angiogenesis are useful for regulating the growth of abnormal blood vessels in diseases such as ARMD, DR and certain retinal degenerations associated with abnormal vasculature.In another preferred embodiment, the Lin" HSCs isolated from the present invention include a gene that encodes a neurotrophic peptide. The Lin "Neurotrophic HSCs are useful for promoting neuronal recovery in ocular diseases involving neural degeneration of the retina, such as glaucoma, retinitis pigmentosa, and the like." A particular advantage of eye treatments with Lin populations "HSCs isolated from The present invention is a vasculotrophic and neurotrophic recovery effect observed in eyes treated intravitreally with the Lin "HSCs." The photoreceptors and neurons of the retina, particularly the cones, are preserved and to some extent the visual function can be maintained in the cells. eyes treated with Lin "HSCs isolated from the invention. Preferably the diseased retina to be treated by the methods of the invention includes activated astrocytes. This can be done by the early treatment of the eye when there is an associated gliosis, or by using a laser to stimulate the local proliferation of activated astrocytes. BRIEF DESCRIPTION OF THE FIGURES Figure 1 depicts schematic diagrams of developing mouse retina, (a) Primary plexus development, (b) The second phase of retinal vessel formation. GCL, ganglion cell layer; IPL, internal layer of plexus; INL nuclear inner layer; OPL, external layer of plexus; ONL, nuclear outer layer; RPE, retinal pigment epithelium; ON, optic nerve; P, periphery. Panel (c) represents the cytometric characterization of the flow of the separated Lin + HSC and Lin cells "HSC derived from the bone marrow Upper row: Distribution in diagram of points of marked cells that are not antibodies, in which Rl defines the area quantifiable interrupted positive PE stain; R2 indicates positive GFP; Middle row: Lin "HSC (C57B / 6) and Bottom row: Lin + HSC cells (C57B / 6), each cell line labeled with PE conjugated antibodies for Sca-1, c-kit, Flk-1 / KDR, CD31. The Tie-2 data were obtained from Tie-2-GFP mice. The percentages indicate the percentage of cells marked as positive outside the total population of Lin "HSC or Lin + HSC." Figure 2 depicts a graft of Lin "HSCs in the developing mouse retina, (a) at four days after the injection (P6) were injected in intravitreal Lin cells "HSC of GFP + e bound and differentiated in the retina (b) Lin" HSC (B6.129S7-Gross26 mice, stained with β-gal antibody) are established in advance of the vasculature stained with collagen IV antibody ( the asterisk indicates the tip of the vasculature), (c) Most Lin + HSC cells (GFP + e) at four days after injection (P6) were unable to differentiate, (d) murine GFP + EC mesenteric four days after the injection (P6). (e) Lin "HSCs (GFP + e) injected into adult mouse eyes, (f) Lin minimal magnification" GFP + e HSCs (arrows) that target and differentiate along the pre-existing astrocytic template in the GFAP-GFP transgenic mouse. (g) Maximum magnification of the association between Lin cells "(GFPe) and the implicit astrocytes (arrows), (h) Transgenic control of non-injected GFAP-GFP, (i) Four days after the injection (P6), the Lin "HSCs of GFP + e migrate to and undergo differentiation in the future deep plexus area.The left figure captures the activity of Lín ~ HSC in a fully assembled retina, the right figure indicates the placement of the Lin cells "(arrows) in the retina (the upper side is the vitreous, the lower side is the sclera), (j) Double labeling with 488 a-CD31-PE and a-GFP-alexa antibodies. Seven days after the injection, the Lin "HSCs (GFPe), red) were incorporated in the vasculature (CD31). The arrowheads indicate the incorporated areas, (k) Lin cells "HSC of GFP + e form the vessels fourteen days after the injection (P17). (1 and m) Intracardiac injection of rhodamine-dextran indicates that the vessels They are intact and functional in both primary (I) and deep plexus (m) Figure 3 shows that Lin cells "HSC of GFP + e are directed to gliosis (indicated by astrocytes that express GFAP, far left image) induced by both laser (a) and mechanical (b) damage induced in the adult retina (the asterisk indicates the damaged site). The right right images are a maximum magnification, showing the close association of the Lin ~ HSCs and the astrocytes. Calibration bar = 20μM. Figure 4 shows that Lin cells "HSC recover the vasculature of the mouse with retinal degeneration, (ad) Retinas at 27 days after injection (P33) with collagen IV staining, (a) and (b), injected retinas with Lin + HSC cells (Balb / c) showed no differences in the vasculature of the normal FVB mice; (c) and (d) retinas injected with Lin "HSCs (Balb / c) showed a rich vascular network analogous to a wild type mouse; (a) and (c), frozen sections of the entire retina (upper is the vitreous side, lower is the sclera side) with DAPI staining; (b) and (d), deep plexus of quantity complete of the retina; (e) bar graph illustrating the increase in vascularity of the vascular deep plexus formed in the retinas injected with Lin ~ HSC cells (n = 6). The degree of deep retinal vascularization was quantified by calculating the total length of the vessels within each image. The average total lengths of the vessels / high power field (in microns) was compared for Lin "HSC, Lin + HSC or control retinas, (f) comparison of the length of the deep vascular plexus after injection with Lin cells" HSC (right eye, D) or Lin + HSC (left eye, I) mouse rd / rd. The results of six independent mice are shown (each color represents each mouse), (g) and (h) Lin cells "HSC also (Balb / c) recovered the rd / rd vasculature when injected into eyes at P15. Intermediate and deep retinas injected with Lin cell "HSC (G) or Lin + HSC (H) (one month after injection) are shown. Figure 5 depicts microphotographs of mouse retinal tissue: (a) deep layer of the complete retina assembly (mouse rd / rd), five days after injection (Pll) with Lin "visible GFP + HSCs (gray). (B) and (c) P60 retinal vasculature of Tie-2-GFP mice (rd / rd) that received Balb / c Lin cells injection" (b) ) or cellsc) to P6. Only endogenous endothelial cells (stained with GFP) are visible in the panels left of (b) and (c). The intermediate panels of (b) and (c) are stained with CD31 antibody; the arrows indicate the vessels stained with CD31 but not with GFP, the right panels of (b) and (c) show staining as much as GFP as CD31. (d) a-SMA staining of Lin ~ HSC injected (left panel) and control retina (right panel). Figure 6 shows that Lin ~ HSCs transfected with T2-TrpRS inhibit the development of mouse retinal vasculature, (a) Schematic representation of TrpRS, T2-TrpRS and human T2-TrpRS with an Igk signal sequence at the amino terminal , (b) the retinas injected with Lin " HSC transfected with T2-TrpRS express the T2-TrpRS protein in vivo. (1) Recombinant T2-TrpRS produced in E. coli; (2) Recombinant T2-TrpRS produced in E. coli; (3) Recombinant T2-TrpRS produced in E. coli; (4) control retina; (5) retina injected with Lin "HSC + pSecTAG_2_A (single vector); (6) retina injected with Lin "HSC + pKLel35 (Igk-T2-TrpRS in Marca pSec), (a) Endogenous TrpRS, (b) T2-Recombinant TrpRS. (c) Retin T2-TrpRS injected with Lin "HSC, (cf) Primary (surface) and secondary (deep) representative plexuses of injected retinas, seven days after injection; (c) and (d) Eyes injected with Lin ~ HSC transfected with an empty plasmid developed normally; (e) and (f) most of the eyes injected with Lin "HSC transfecta.do with T2-TrpRS showed inhibition of the deep plexus; (c) and (e) primary (surface) plexus; (d) and (f) secondary (deep) plexus. Mild tracing of the vessels observed in (f) are images of "exudate" from the vessels of the primary network shown in (e). Figure 7 shows the DNA sequence encoding His6-tagged T2-TrpRS, SEQ ID NO: 1. Figure 8 shows the amino acid sequence of His6-tagged T2-TrpRS, SEQ ID NO: 2. Figure 9 illustrates microphotographs and electroretinograms (ERG) of retinas of mice whose eyes were injected with the Lin'HSCs of the present invention and with Lin + HSC (controls). Figure 10 depicts statistical graphs showing a correlation between neuronal recovery (y-axis) and vascular recovery (x-axis) for both intermediary (Int.) Vascular layers and deep rd / rd mouse eyes treated with Lin "HSC Figure 11 represents statistical graphs showing no correlation between neuronal recovery (y-axis) and vascular recovery (x-axis) for rd / rd mouse eyes that were treated with Lin + HSC Figure 12 is a bar graph vascular length (y-axis) in relatively arbitrary units for rd / rd mouse eyes treated with Lin "HSC (dark bars) and untreated rd / rd mouse eyes (light bars) at points of time of 1 month (IM), 2 months (2M), and 6 months (6M) after the injection. Figure 13 includes three bar graphs of the number of nuclei in the outer neural layer (ONR) of rd / rd mice in 1 month (IM), 2 months (2M) and 6 months (6M), after injection, and shows a significant increase in the number of nuclei for eyes treated with Lin "HSC (dark bars) with respect to the control eyes treated with Lin + HSC (light bars) Figure 14 represents graphs of the number of nuclei in the external neural layer for individual rd / rd mice, comparing the right eye (D, treated with Lin "HSC) with respect to the left eye (I, control eye treated with Lin + HSC) at time points (post-injection) of 1 month (1M) , 2 months (2M) and 6 months (6M); Each line in a given graph compares the eyes of an individual mouse. Figure 15 depicts retinal vasculature and neural cell changes in rdl / rdl (C3H / HeJ, left panels) or wild type (C57BL / 6, right panels) mice. The retinal vasculature of the intermediate vascular plexus (upper panels) or deep plexus (intermediate panels) in the completely mounted retinas (red: collagen IV, green, CD31) and the sections (red: DAPI, green: CD31, lower panels) of the The same retinas are shown (P: postnatal day). (GCL: cellular layer of ganglion, INL: internal nuclear layer, ONL, outer nuclear layer). Figure 16 shows that the injection of Lin "HSC recovers the degeneration of neural cells in rdl / rdl mice (A, B and C), the retinal vasculature of the intermediate (int.) Or deep plexus and the sections of the injected eye with Lin "HSC (right panels) and eyes injected with collateral control cell (CD31") (left panels) in P30 (A), P60 (B), and P180 (C). (D), the average total length of the vasculature (+ or - standard error of the average) in retinas injected with Lin "HSC or injected with the control cell (CD31") in P30 (left, n = 10), P60 (intermediate, n = 10), and P180 (right, n = 6) The intermediate (int.) and deep vascular plexus data are shown separately (Y axis: relative length of the vasculature). (E), the average numbers of cell nuclei in the ONL in P30 (left, n = 10), P60 (medium, n = 10), or P180 (right, n = 6) of retinas injected with control cell CD31") or with Lin" HSC (Y axis: relative number of nucleus s cell phones in the ONL). (F), linear correlations between the length of the vasculature (X axis) and the number of cell nuclei in the ONL (Y axis) in P30 (left), P60 (middle), and P180 (right) of retinas injected with Lin "HSC or with control cell." Figure 17 shows that the function of the retina is recovered by injection of Lin "HSC." Electroretinographic (ERG) records were used to measure the function of retinas injected with Lin "HSC or with control cell (CD31"). (A and B), Representative cases of recovered retinas and not recovered 2 months after the injection The retinal section of the right eye injected with Lin "HSC (A) and the left eye injected with control cell CD31" (B) of the same animal are shown (green: vasculature stained with CD31, red: nuclei stained with DAPI). (C), ERG results from the same animal shown in (A and B) Figure 18 shows that a population of human bone marrow cells can recover the degenerated retinas in the mouse rdl (AC Recovery is also observed in another model of retinal degeneration, rdl O (D-K). A, human Lin'HSCs (hLin "HSCs) marked with green ink can differentiate into retinal vascular cells after intravitreal injection in SCID mice C3SnSmn. CBll-Prkdc. (B and C), retinal vasculature (left panels, upper: intermediate plexus, lower: deep plexus) and neural cells (right panel) in eyes injected with hLin "HSC (B) or contralateral control eye (C) 1.5 months after injection (DK), Recovery of rdl O mice by Lin "HSCs (injected in P6). Representative retinas are shown in P21 (D: Lin "HSCs, H: cells control), P30 (E: Lin "HSCs, I: control cells), P60 (F: Lin" HSCs, J: control cells), and P105 (G: Lin "HSCs, K: control cells) (eyes treated and control are from the same animal at each time point.) The retinal vasculature (the upper image on each panel is the intermediate plexus, the average image on each panel is the deep plexus) was stained with CD31 (green) and Collagen IV ( red) The lower image on each panel shows a cross section made of the same retina (red: DAPI, green: CD31) Figure 19 shows that the crystalline aA is up regulated in the outer nuclear layer cells recovered after treatment with Lin "HSCs but not in contralateral eyes treated with control cells. Left panel; Control IgG in retina recovered, Medium panel; crystalline aA in recovered retina, right panel; crystalline aA in retina not recovered. Figure 20 includes gene tables that are up-regulated in murine retinas that have been treated with the Lin "HSCs of the present invention. (A) Genes whose expression is increased 3-fold in mouse retinas treated with Lin" HSCs of murine (B) Crystalline genes that are over-regulated in mouse retinas treated with Lin "murine HSC. (C) Genes whose expression is increased 2-fold in mouse retinas treated with Lin" Human HSCs. (D) Genes for neurotrophic factors or growth factors whose expression is up-regulated in mouse retinas treated with Lin "human HSCs." Figure 21 illustrates the distribution of surface antigens of CD31 and integrin a6 in populations of Lin "human HSC CD133 positive (DC133 +) and negative CD133 (CD133") of the present invention The left panels show scattered flow cytometric plots The central and right panels are histograms showing the level of expression of specific antibody in the cell population The Y axis represents the number of events and the X axis shows the intensity of the signal A full histogram shifted to the right of the plotted histogram (control) represents an increased fluorescent signal and the expression of the antibody above the basic level Figure 22 illustrates the development of the postnatal retina in wild type C57 / B16 mice produced at normal oxygen levels (normoxia), on postnatal days PO to P30 Figure 23 illustrates a model of oxygen-induced retinopathy in C57 / B16 mice produced at high oxygen levels (hyperoxia; 75% oxygen) between P7 and P12, followed by normoxia of P12-P17 Figure 24 shows vascular recovery by treatment with the population Lin "HSC of this invention in the model of oxygen-induced retinopathy. Figure 25 shows the photoreceptors recovered in the outer nuclear layer (ONL) of an rdl mouse that after the intravitreal injection of Lin "HSC are predominantly cones." A small percentage of the photoreceptors in the wild type mouse retina (upper panel) were cones as evidenced by the red / green cone opsin expression (A) while the majority of the ONL cells were positive for rod-specific rhodopsin. (B) The retinal vasculature auto-fluoresces with pre-immune serum (C) but the nuclear layers were completely negative for cone or rod-specific opsin staining.Rd / rd mouse retinas (lower panels) had a decreased internal nuclear layer and an almost completely atrophic ONL, both of which were Negative for opsin of cones (D) or sticks (Panel G) Control eyes treated with CD31"HSC are identical to retinas rd / rd not injected, without any staining for cone opsina (E) or cane (H). The contralateral eyes treated with Lin ~ HSC showed a markedly reduced ONL, but clearly present that is predominantly comprised of cones, as evidenced by positive immunoreactivity for opsin of red / green cones (F). A small number of canes were also observed (I).

DETAILED DESCRIPTION OF THE INVENTION Stem cells are typically identified by the distribution of antigens on the surface of cells (for a detailed discussion see Stem Cells: Scientific Progress and Future Directions, a report prepared by the National Institutes of Health, Office of Science Policy , June 2001, Appendix E: Stem Cell Markers, which is incorporated herein by reference to the relevant grade). The present invention provides a method for alleviating cellular degeneration of cones in the retina of a mammal suffering from an eye disease. A population of isolated, negative lineage hematopoietic stem cells derived from mammalian bone marrow, comprising 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 alleviate cellular degeneration of cones in the retina. A preferred method comprises isolating the population of hematopoietic stem cells, of negative lineage, from the bone marrow of the mammal to be treated and subsequently administering the cells to the mammal in a sufficient number to alleviate cellular degeneration of cones in the retina.

The cells can be obtained from the diseased mammal, preferably at an early stage of the ocular disease. Alternatively, the cells can be obtained before the onset of the disease in a patient known to have a genetic predisposition to an ocular disease such as retinitis pigmentosa, for example. The cells can be stored until they are needed, and can then be prophylactically injected at the first signal observed at the onset of the disease. Preferably the diseased retina includes activated astrocytes, to which the stem cells are directed. Accordingly, the early treatment of the eye when there is an associated gliosis is beneficial. Alternatively, the retina can be treated with a laser to stimulate the local proliferation of activated astrocytes in the retina before administering the autologous stem cells. Hematopoietic stem cells are those stem cells that are capable of developing into different types of blood cells e. g. , B cells, T cells, granulocytes, platelets and erythrocytes. Lineage surface antigens are a group of surface proteins in the cell that are markers of lineages of mature blood-blood cells, including CD2, CD3, CD11, CDlla, Mac-1 (CDllb: CD18), CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, CD45RA, murine Ly-6G, murine TER-119, CD56, CD64, CD68, CD86 (B7.2), CD66b, antigen human leukocyte DR (HLA-DR), and CD235a (Glycoforin A). Hematopoietic stem cells that do not express significant levels of these antigens are commonly referred to as a negative lineage (Lin ".) Human hematopoietic stem cells commonly express other surface antigens such as CD31, CD34, CD117 (c-kit) and / or CD133. The murine haematopoietic stem cells commonly express other surface antigens such as CD34, CD17 (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 its cell surfaces Such cells are referred to herein as "negative lineage" or Lin "hematopoietic stem cells. In particular this invention provides a population of Lin hematopoietic stem cells (Lin ~ HSCs) which includes endothelial progenitor cells (EPCs), which are capable of being incorporated into the developing vasculature and then differentiated into vascular endothelial cells. of Lin "Isolated HSCs are present in a culture medium such as buffered phosphate salt (PBS). As used herein and in the appended claims, the phrase "adult" in reference to bone marrow includes postnatally isolated bone marrow, i. and. , of individuals young and adults, opposed to embryos. The term "adult mammal" refers to both juvenile mammals and fully mature mammals. The isolated, mammals negative lineage (Lin ~ HSC) hematopoietic stem cells of the invention include endothelial progenitor cells (EPCs). Lin populations "isolated HSCs preferably comprise mammalian cells in which at least about 20% of the cells express the CD31 surface antigen, which is commonly present in the endothelial cells." In another embodiment, at least about 50% of the cells express CD31, more preferably at least about 65%, more preferably at least about 75% Preferably at least about 50% of the cells of the Lin "HSC populations of the present invention preferably express the a6 integrin antigen . In a preferred Lin "HSC mouse population, at least about 50% of the cells express CD31 antigen and at least about 50% of the cells express the CD117 antigen (c-kit) .Preferably, at least about 75% of the Lin cells "HSC express the CD31 surface antigen, more preferably approximately 81% of the cells. In another preferred murine embodiment, at least about 65% of the cells express the CD117 surface antigen, more preferably about 70% of the cells. A particularly preferred embodiment of the present invention is a population of Lin "murine HSC in which approximately 50% to approximately 85% of the cells express the CD31 surface antigen and approximately 70% to approximately 75% of the cells express the surface antigen CD117 Another preferred embodiment is a population of Lin "human HSC in which the cells are CD133 negative, in which at least about 50% of the cells express the CD31 surface antigen and at least about 50% of the cells express the integrin 6 antigen. Yet another preferred embodiment is a population of Lin "human HSC in which the cells are CD133 positive, in which less than about 30% of the cells express the CD31 surface antigen and less than about 30% of the cells expressing the a6 integrin antigen The Lin "HSC populations isolated from the present invention are selectively targeted against astrocytes and are incorporated into the retinal neovasculature when they are injected intravitreally into the eye of the mammalian species, such as a mouse or a human, from which the cells were isolated.

Lin populations "HSCs isolated from the present invention include endothelial progenitor cells that differentiate endothelial cells and generate vascular structures within the retina." In particular, the Lin "HSC populations of the present invention are useful for the treatment of retinal vascular degenerative diseases. and neovascular retinal, and to repair a retinal vascular lesion. The "HSC" Lin cells of the present invention also promote neuronal recovery in the retina and promote the up-regulation of anti-apoptotic genes It has been surprisingly found that the "human HSC adult Lin cells of the present invention can inhibit the degeneration of the retina even in mice with severe combined immunodeficiency (SCID) suffering from retinal degeneration. The normal mouse retina is predominantly canes, but the recovered cells, after treatment with Lin "HSC are almost completely cones." Additionally, the Lin populations "HSC can be used to treat retinal defects in the eyes of neonatal mammals, such as mammals suffering from oxygen-induced retinopathy or retinopathy of prematurity. The present invention also provides a method for treating ocular diseases in a mammal that comprises isolating a mammalian bone marrow from a population of hematopoietic stem cells negative lineage including endothelial progenitor cells, and intravitreally inject isolated stem cells in a mammalian eye in a sufficient number to stop the disease. The present method can be used to treat ocular diseases such as retinal degenerative diseases, retinal vascular degenerative diseases, ischemic retinopathies, vascular hemorrhages, vascular effusions and choroidopathies in neonatal, juvenile or fully mature mammals. Examples of such diseases include age-related macular degeneration (ARMD), diabetic retinopathy (DR), suspected ocular histoplasmosis (POHS), retinopathy of prematurity (ROP), sickle cell anemia, and retinitis pigmentosa, as well as retinal injuries. The number of stem cells injected into the eye is sufficient to stop the disease state of the eye. For example, the number of cells can be effective to repair retinal damage of the eye, stabilize retinal neovasculature, mature retinal neovasculature, and prevent or repair spillage or vascular leakage and vascular hemorrhage. The cells of the Lin "HSC populations of the present invention can be transfected with therapeutically useful genes, such as genes encoding anti-angiogenic proteins for use in ocular therapy. of cell-based genes, and genes that code for neurotrophic agents to increase the effects of ocular recovery. The transfected cells can include any gene that is therapeutically useful for the treatment of retinal disorders. In a preferred embodiment, the Lin "transfected HSCs of the present invention include a gene that functionally encodes an anti-angiogenic peptide, including proteins, or protein fragments such as TrpRS or anti-angiogenic fragments thereof, eg, Ti fragments. and T2 of TrpRS, which are described in detail in co-pending US Patent Application Serial No. 10 / 080,839, the disclosure of which is incorporated herein by reference.The Lin "Transfected HSCs that encode an anti-peptide. -angiogenic of the present invention are useful for the treatment of diseases of the retina that involve abnormal vascular development, such as diabetic retinopathy, and similar diseases. Preferably the Lin "HSC's are human cells In another preferred embodiment, the Lin" transfected HSCs of the present invention include a gene that functionally encodes a neurotrophic agent such as nerve growth factor, neurotrophin-3, neurotrophin-4, neurotrophin- 5, ciliary neurotrophic factor, factor neurotrophic retinal pigmented epithelium, insulin-like growth factor, neurotrophic factor derived from the glial cell line, neurotrophic factor derived from the brain, and the like. Such Lin "Neurotrophic HSCs are useful for promoting neuronal recovery in retinal neuronal degenerative diseases such as glaucoma and retinitis pigmentosa, in the treatment of retinal nerve injuries, and the like.The implants of ciliary neurotrophic factor have been reported as useful for the treatment of retinitis pigmentosa (see Kirby et al., 2001, Mol Ther 3 (2): 241-8, Farrar et al., 2002, EMBO Journal 21: 857-864.) Brain derived neurotrophic factor regulates reportedly genes associated with growth in injured retinal ganglia (see Fournier, et al, 1997, J. Neurosci Res. 47: 561-572) The neurotrophic factor derived from the glial cell line reportedly delays photoreceptor degeneration in retinitis pigmentosa (see McGee et al 2001, Mol Ther 4 (6): 622-9) The present invention also provides a method for isolating negative lineage hematopoietic stem cells comprising cells endothelial progenitor bone marrow of a mammal. 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) label the monocytes with a panel of lineage antibodies conjugated with biotin to one or more lineage surface antigens, preferably lineage surface antigens selected from the group consisting of CD2, CD3, CD4, CDll, CDlla, Mac- 1, CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, Ly-6G (murine), TER-119 (murine), CD45RA, CD56, CD64, CD68, CD86 (B7.2), CD66b, antigen of human leukocyte DR (HLA-DR), and CD235a (Glycoforin A); (d) removing monocytes that are positive for said one or more lineage surface antigens from the plurality of monocytes and recovering a population of negative lineage hematopoietic stem cells containing endothelial progenitor cells, preferably, at least about 20% of the cells express CD31. When the Lin "HSCs are isolated from the bone marrow of an adult human, preferably monocytes are labeled with a panel of lineage antibodies conjugated with biotin to the surface antigens of lineage CD2, CD3, CD4, CDlla, Mac-1, CD14 , CD16, CD19, CD33, CD38, CD45RA, CD64, CD68, CD86, CD86 (B7.2), and CD235a When Lin "HSCs are isolated from the bone marrow of adult murine, preferably monocytes are labeled with a panel of antibodies. of lineage conjugated with biotin to surface antigens of lineage CD3, CD11, CD45, Ly-6G, and TER-119. In a preferred method, the cells are isolated from adult human bone marrow and are further separated by the CD133 lineage. A preferred method for isolating Lin "human HSCs includes the additional steps of labeling monocytes with a CD133 antibody conjugated with biotin and recovering a population of Lin" HSC, CD133 positive. Typically, less than about 30% of such cells express CD31 and less than about 30% of such cells express integrin a6. Lin populations "human, CD133 positive HSCs of the present invention can recognize sites of neovascularization driven by peripheral ischemia when injected into eyes that do not undergo angiogenesis Another preferred method to isolate Lin" Human HSCs include the additional steps of labeling monocytes with a CD133 antibody conjugated with biotin, remove CD133 positive cells, and recover a Lin population "negative CD133 HSC." Typically, at least about 50% of such cells express CD31 and at least about 50% of such cells express integrin a6. Lin populations "human, CD133 negative HSCs of the present invention can be incorporated into developing vasculature when injected into eyes suffering from angiogenesis.

The present invention also provides methods for treating ocular angiogenic diseases by administering Lin cells "transfected HSCs of the present invention by intravitreal injection of the cells in the eye." Such Lin cells "transfected HSCs comprise Lin" HSCs transfected with a therapeutically useful gene, such as a gene encoding a product of the antiangiogenic or neurotrophic gene Preferably the Lin cells "transfected HSCs are human cells. Preferably, at least about 1 × 10 5 Lin cells "HSC or Lin cells" transfected HSC are administered by intravitreal injection to a mammalian eye suffering from a retinal degenerative disease. The number of cells to be injected may depend on the severity of the retinal degeneration, the age of the mammal and other factors that will be readily apparent to one skilled in the art of treating retinal diseases. The Lin "HSC can be administered in a single dose or by administering multiple doses for a period of time, as determined by the treating physician.The Lin" HSCs of the present invention are useful for the treatment of damage retinal and retinal defects that involve an interruption in or degradation of the retinal vasculature or neuronal retinal degeneration. The Lin "Human HSCs can also be used to generate a line of genetically identical cells, ie, clones, for use in a regenerative or reparative treatment of the retinal vasculature, as well as to treat or alleviate retinal neuronal degeneration." EXAMPLES Example 1. Enrichment and Cellular Isolation: Preparation of Populations A and B of Lin "Murine HSC General Procedure. All in vivo evaluations were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and all evaluation procedures were approved by the Animal Use and Care Committee of The Scripps Research Institute (TSRI, La Jolla , CA). Bone marrow cells were extracted from B6.129S7-Gross26, Tie-2GFP, ACTbEGFP, FVB / NJ mice (rd / rd mice) or adult mice Balb / cBYJ (The Jackson Laboratory, ME). The monocytes were then separated by density gradient separation using HISTOPAQUE® polysucrose gradient (Sigma, St. Louis, MO) and labeled with biotin-conjugated lineage panel antibodies.

(CD45, CD3, Ly-6G, CD11, TER-119, Pharmingen, San Diego, CA) for the Lin selection "in mice. positive lineage (Lin +) were separated and removed from Lin "HSC using a magnetic separation device (AUTOMACSMR classifier, Miltenyi Biotech, Auburn, CA). The resulting Lin "HSC population, which contained endothelial progenitor cells was further characterized using a FACSMR Calibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ) using the following antibodies: PE-conjugate-Sca-1, c-kit, KDR, and CD31 (Pharmingen, San Diego, CA) Bone marrow cells Tie-2-GFP were used for the characterization of Tie-2 To harvest adult mouse endothelial cells, the mesenteric tissue was surgically removed from an ACTbEGFP mouse and placed in collagenase (Worthington, Lakewood, NJ) to digest the tissue, followed by filtration using a 45μm filter.The flow was collected and incubated with Endothelial Growth Medium (Clonetics, San Diego, CA) .The endothelial characteristics were confirmed. observing the morphological appearance of cobblestone, staining with CD31 mAb (Pharmingen) and examining the cultures for the formation of tube-like structures in the MATRIGELMR matrix (Beckton Dec kinson, Franklin Lakes, NJ). Population A of Lin "HSC Murina Bone marrow cells were extracted from ACTbEGFP mice by the General Procedure described previously. The Lin "HSC cells were characterized by FACS flow cytometry for the markers of cell surface antigens CD31, c-kit, Sca-1, Flk-1, and Tie-2. The results are shown in Figure 1 (c). Approximately 81% of Lin "HSC showed the CD31 marker, approximately 70.5% of Lin-HSC showed the c-kit marker, approximately 4% of Lin-HSC showed the Sca-1 marker, approximately 2.2% of Lin-HSC showed the marker Flk-1 and approximately 0.91% Lin-HSC showed the Tie-2 marker. In contrast, the Lin + HSCs that were isolated from these bone marrow cells had a significantly different cell marker profile (ie, CD31: 37.4%, c-kit: 20%, Sca-1: 2.8%, Flk-: 0.05%) . Population B of Lin "HSC Murina Bone marrow cells were extracted from Balb / C mice, ACTbEGFP, and C3H by the General Procedure described above Lin cells" HSC were analyzed for the presence of cell surface markers (Sca-1 , KDR, c-kit, CD34, CD31 and different integrins: al, a2, a3, a4, a5, a6, aM, av, ax, am ,, ßi, ß, ß3 / - ß4r ßs and ßv). The results are shown in Table 1.

Table 1 . Characterization of Population B of Lin ~ HSC, Cellular Marker Lin "HSC at 0.10 a2 17.57 a3 0.22 a4 89.39 a5 82.47 a6 77.70 a 62.69 aM 35.84 aX 3.98 aV 33.64 allb 0.25 ßl 86.26 ß2 49.07 ß3 45.70 ß4 0.68 ß5 9.44 ß7 11.25 CD31 51.76 CD34 55.83 Flk-1 / KDR 2.95 c -kit (CD117) 74.42 Sca-1 7.54 Example 2. Intravitreal Administration of Cells in a Murine Model An eyelid fissure was created in a mouse eyelid with a thin knife to expose the eyeball from P2 to P6. The HSC Population A of negative lineage of the present invention (about 105 cells in about 0.5 μl to about 1 μl of cell culture medium) was then injected intravitreally using a 33 gauge syringe needle (Hamilton, Reno, NV) . Example 3. Transfection of the EPC. Lin "Murine HSCs (Population A) were transfected with DNA encoding the T2 fragment of TrpRS that also includes a His6 tag (SEQ ID NO: 1, FIG.7) using the transfection reagent FuGENEMR6 (Roche, Indianapolis, IN) according to with the manufacturer's protocol Lin cells "HSC (approximately 10 6 cells per ml) were suspended in opti-MEM® medium (Invitrogen, Carlsbad, CA) containing the stem cell factor (PeproTeeh, Rocky Hill, NJ). After adding a mixture of DNA (approximately 1 μg) and FuGENE reagent (approximately 3 μl), and the mixtures were incubated at approximately 37 ° C for approximately 18 hours. After incubation, the cells were washed and harvested. The transfection rate of this system was approximately 17% which was confirmed by FACS analysis. The production of T2 was confirmed by western blotting. The amino acid sequence of T2-TrpRS labeled with Hise is shown as SEQ ID NO: 2, FIG. 8 Example 4. Immunohistochemistry and Confocal Analysis. Mouse retinas were harvested at different time points and were prepared either for freeze-off or complete assembly. For complete assemblies, the retinas were fixed with 4% paraformaldehyde, and blocked in 50% fetal bovine serum (FBS) and 20% normal goat serum for one hour at room temperature. The retinas were processed by primary antibodies and detected with secondary antibodies. The primaries used were: anti-Collagen IV (Chemicon, Temecula, CA), anti-ß-gal (Promega, Madison, Wl), anti-GFAP (Dako Cytomation, Carpenteria, CA), anti-a smooth muscle actin ( a-SMA, Dako Cytomation). Secondary antibodies used were conjugated to either fluorescent markers Alexa 488 or 594 (Molecular Probes, Eugene, OR). The images were taken using a MRC 1024 Confocal microscope (Bio-Rad, Hercules, C?). Three-dimensional images were created using the LASERSHARP® program (Bio-Rad) to examine the three different layers of vascular development in the fully assembled retina. The difference in GFP pixel intensity between augmented GFP mice (eGFP) and GFAP / wtGFP mice, distinguished by confocal microscopy, was used to create the 3-dimensional images. Example 5. Retinal Angiogenesis Quantification Assay in Live in Mice. For T2-TrpRS analysis, the primary and deep plexuses were reconstructed from three-dimensional images of mouse retinas. The primary plexus was divided into two categories: normal development, or interrupted vascular evolution. The categories of inhibition of deep vascular development were constructed based on the percentage of vascular inhibition including the following criteria: complete inhibition of deep plexus formation was marked "Complete", normal vascular development (including less than 25% inhibition) it was marked "Normal" and the rest marked "Partial". For the rd / rd mouse recovery data, four separate areas of the deepest plexus were captured in each fully mounted retina using an lOx lens. The total length of the vasculature was calculated for each image, summed and compared between the groups. For precise information, Lin "HSC was injected into one eye and Lin + HSC into the other eye of the same mouse." Non-injected control retinas were taken from the same litter .. Example 6. Murine Models with Adult Retinal Lesion. and lasers were created using a diode laser (150 W, 1 second, 50 mm) or mechanically puncturing the mouse's retina with a 27-gauge needle. Five days after the damage, the cells were injected using the intravitreous method. The eyes were harvested from the mice five days later. Example 7. Rhenic Neuro Recovery of Retinal Regeneration.

Negative line hematopoietic stem cells (Lin ~ HSC) derived from adult murine bone marrow have a vasculotrophic and neurotrophic recovery effect in a mouse model of retinal degeneration. The right eyes of 10-day-old mice were injected intravitreally with approximately 0.5 microliters containing approximately 105 Lin "HSC of the present invention and evaluated 2 months later for the presence of retinal vasculature and nuclear counting of the neuronal layer. Left eyes of the same mice were injected with approximately the same number of Lin + HSC as a control, and were evaluated in a similar manner.As shown in Figure 9, in eyes treated with Lin "HSC, the retinal vasculature seemed almost normal , the inner nuclear layer was almost normal and the outer nuclear layer (ONL) had approximately 3 to approximately 4 layers of nuclei. In contrast, the eye treated with Lin + contralateral HSC had a markedly atrophic medial vascular layer, a completely atrophic external retinal vascular layer; the inner nuclear layer was markedly atrophic and the outer nuclear layer was completely ruined. This was dramatically illustrated in Mouse 3 and Mouse 5. In Mouse 1 there was no recovery effect and this was true for approximately 15% of the mice injected. When the visual function was evaluated with electroretinograms (ERG), the restoration of a positive ERG was observed when both of the vascular and neuronal recoveries were observed (Mice 3 and 5). The positive ERG was not observed when there was no vascular and neuronal recovery (Mouse 1). This correlation between the vascular and neurotrophic recovery of the mouse eyes rd / rd by the Lin "HSC of the present invention is illustrated by a graph of regression analysis shown in Figure 10. A correlation between neuronal recovery (y axis) and vascular (x-axis) was observed for the intermediate vasculature type (r = 0.45) and for the deep vasculature (r = 0.67) Figure 11 shows the absence of any statistically significant correlation between vascular and neuronal recovery by Lin + HSC The vascular recovery was quantified and the data are presented in Figure 12. The data for the mice in 1 month (IM), 2 months (2M), and 6 months (6M), after the injection shown in Figure 12. , show that the vascular length was significantly increased in eyes treated with the Lin "HSC of the present invention (dark bars) with respect to the vascular length in untreated eyes of the same mouse (clear bars), particularly in 1 month and 2 months after injection. The effect of neurotrophic recovery was quantified by counting the nuclei in the inner and outer nuclear layers approximately two months after the Lin injection "HSC or Lin + HSC." The results are presented in Figures 13 and 14. Example 8. Population Lin " HSC Human. Bone marrow cells were extracted from healthy adult human volunteers by the General Procedure described above. The monocytes were then separated by density gradient separation using HISTOPAQUE® polysucrose gradient (Sigma, St. Louis, MO). To isolate the Lin "HSC population of human bone marrow mononuclear cells, the following lineage panel antibodies conjugated with biotin were used with the magnetic separation system.

(AUTOMACSMR classifier, Miltenyi Biotech, Auburn, CA): CD2, CD3, CD4, CDlla, Mac-1, CD14, - CD16, CD19, CD33, CD38, CD45RA, CD64, CD68, CD86, CD235a (Pharmingen). The Lin population "human HSC was further separated into two sub-populations based on CD133 expression." The cells were labeled with CD133 antibodies conjugated with biotin and separated into CD133 positive and CD133 negative subpopulations.

Example 9. Intravitreal Administration of Human and Murine Cells in Murine Models for Retinal Degeneration. Mouse strains C3H / HeJ, C3SnSmn.CB17-Prkdc SCID, and rdl O were used as models of retinal degeneration. The C3H / HeJ and C3SnSmn.CB17-Pr ^ dc SCID mice (The Jackson Laboratory, Maine) were homozygous for the mutation 1 (rdl) of retinal degeneration, a mutation that causes severe early-onset retinal degeneration. The mutation is located in exon 7 of the Pdedb gene that encodes the β subunit of the phosphodiesterase of the photoreceptor cGMP of rods. The mutation in this gene has been found in human patients with autosomal recessive retinitis pigmentosa (RP). C3SnSmn.CB17-PrA "dc SCID mice are also homozygotes for the spontaneous mutation of severe combined immunodeficiency (Prkdc SCID) and were used for human cell transfer experiments.Retinal degeneration in rdl O mice is caused by a mutation in the exon 13 of the pdeßb gene This is also a clinically relevant RP model with more moderate retinal degeneration and initiated later than rdl / rdl.) All evaluations were performed according to the Guide for the Care and Use of Laboratory Animals, and All procedures were approved by the Animal Use and Care Committee of The Scripps Research Institute, an eyelid fissure was created on the eyelid of a mouse with a thin knife to expose the eyeball from P2 to P6. HSC cells of negative lineage for murine Population A or Human Population C (approximately 105 cells in approximately 0.5 μl to approximately 1 μl of cell culture medium) were then injected into the mouse eye intravitreally using a gauge syringe needle 33 (Hamilton, Reno, NV). To visualize the injected human cells, the cells were marked with ink (green cell signaling CMFDA, Molecular Probes) before injection. The retinas were harvested at various time points and fixed with 4% paraformaldehyde (PFA) and methanol followed by blocking in 50% FBS / 20% NGS for one hour at room temperature. To stain the retinal vasculature, the retinas were incubated with anti-CD31 (Pharmingen) and anti-collagen IV (Chemicon) antibodies followed by Alexa 488 or 594 conjugated secondary antibodies (Molecular Probes, Eugene, Oregon). The retinas were placed extended with four soothing radial incisions to obtain a complete assembly preparation. Images of the vasculature in the intermediate or deep retinal vascular plexus (see, Dorrell et al, 2002 Jnvest Ophthalmol, Sci 43: 3500-3510) were obtained using a Radiance MP2100 confocal microscope and the LASERSHARP® program (Biorad, Hercules , California). For quantification of vasculature, four independent fields (900 μm x 900 μm) were randomly selected from the middle portion of the intermediate or deep vascular layer and the total length of the vasculature was measured using the LASERPIX® analysis program (Biorad) . The total lengths of these four fields in the same plexus were used for further analysis. The extended-mounted retinas were re-imbedded for cryostat sections. The retinas were placed in 4% PFA overnight followed by incubation with 20% sucrose. The retinas were embedded in an optimal cutting temperature compound (OCT: Tek-Tek, Sakura FineTech, Torrance, C?). The cryostatic sections (10 μm) were re-hydrated in PBS containing the DAPI nuclear ink (Sigma-Aldrich, St. Louis, Missouri). The nuclear images marked with DAPI from three different areas (280 μm wide, neutral sampling) in a single section containing the head of the optic nerve and the entire peripheral retina were taken by a confocal microscope. The number of nuclei located in the ONL of the three independent fields in a section were counted and added for analysis. A simple linear regression analysis was performed to examine the relationship between the length of the vasculature in the deep plexus and the number of cell nuclei in the ONL.

After they were adapted to darkness overnight, the mice were anesthetized by intraperitoneal injection of 15 μg / g of ketamine and 7 μg / g of xylazine. Electroretinograms (ERGs) were recorded from the corneal surface of each eye after dilatation of the pupil (1% atropine sulfate) using a gold-plated corneal electrode together with a reference electrode in the mouth and ground in the tail. The stimuli were produced with a Grass Fotic stimulator (PS33 Plus, Grass Instruments, Quincy, MA) fixed to the outside of a highly reflective Ganzfel dome. Rod responses were recorded at light flashes with short wavelength (Wratten 47A;? Max = 470 nm) with respect to a range of intensities up to the maximum allowable by the photic stimulator (0.668 cd-s / m2). The response signals were amplified (AC amplifier CP511, Grass Instruments), digitized (PCI-1200, National Instruments, Austin, TX) and analyzed by computer. Each mouse served as its own internal control with registered ERGs of both treated and untreated eyes. Up to 100 sweeps were averaged for the weakest signals. The averaged responses of the untreated eye were digitally subtracted from the responses of the treated eye and this difference in signal was used to index functional recovery.

Microarray analysis was used to evaluate the expression of the retinal target gene recognized from Lin ~ HSC. P6 rd / rd mice were injected with Lin "or CD31" HSCs. The retinas of these mice were sectioned 40 days after injection in RNase-free medium (the recovery of the retinal vasculature and the photoreceptor layer is obvious at this time point after the injection). One quadrant of each retina was analyzed by complete assembly to ensure that the normal target of HSC as well as the vasculature and neural protection had been achieved. The retinal RNA with successful injections was purified using TRIzol (Life Technologies, Rockville, MD), RNA isolation protocol with phenol / chloroform. The RNA was hybridized to Affymetrix Mu74Av2 chips and the expression of the gene was analyzed using the GENESPRING® program (SiliconGenetics, Redwood City, CA). Purified HSCs from human or mouse were injected intravitreally into P6 mice. In P45 the retinas were dissected and pooled into fractions of 1) recovered mouse retinas, injected with human HSCs, 2) unrecovered mouse retinas, injected with human HSC, and 3) recovered mouse retinas, injected with mouse HSC for the purification of RNA and hybridization to human-specific Affymetrix U133A chips. The GENESPRING® program was used to identify the genes that were expressed above the background and with greater expression in the retinas recovered with human HSC. The expression profiles of the pair of assays for each of these genes were then analyzed individually and compared with a model of normal human U133A microarray experiments using Chip-d to determine the specific hybridization to human species and to eliminate false positives due to Hybridization of crossed species. Figure 21 illustrates the flow cytometry data comparing the expression of the CD31 and integrin a6 surface antigens in Lin populations "CD133 positive (CD133 +) and CD133 negative (CD133") human HSCs of the present invention, the left panels show the scattered graphs of flow cytometry. The center and right panels are histograms that show the level of expression of the specific antibody in the cell population. The Y axis represents the number of events and the X axis shows the intensity of the signal. The plotted histograms are IgG isotype control antibodies that show the non-specific basic staining level. The full histograms show the level of expression of the specific antibody in the cell population. A full histogram shifted to the right of the plotted (control) histogram represents a fluorescent signal increased and antibody expression above the basic level. Comparing the position of the peaks of the filled histograms between the two cell populations represents the difference in the expression of the protein in the cells. For example, CD31 was expressed above the background in both CD133 + and CD133- cells of the invention; however, there are more cells that express lower levels of CD31 in the CD133 + cell population than there is in the CD133 population. "From these data it is evident that the expression of CD31 varies between the two populations and that the expression of integrin a6 it is very limited for cells in the Lin population, and therefore can serve as a marker of cells with function of vascular and neurotrophic recovery. When the Lin sub-population "HSC CD133 positive and CD 133 negative were injected intravitreally into the eyes of neonatal SCID mice, the largest degree of incorporation into the developing vasculature was observed for the negative CD133 sub-population, which it expresses both surface antigens CD31 and integrin a6 (see Figure 21, bottom) The positive CD133 sub-population, which does not express CD31 or integrin a6 (Figure 21, upper) appears for target sites of neovascularization directed by peripheral ischemia, but does not when injected into eyes suffering from angiogenesis.

The recovered and unrecovered retinas were analyzed immunohistochemically with antibodies specific for opsin from rods or cones. The same eyes used for the ERG records presented in Figure 17 were analyzed for opsin of rods or cones. In wild-type mouse retinas, less than 5% of the photoreceptors present are cones (Soucy et al., 1998, Neuron 21: 481-493) and the immunohistochemical staining patterns observed with opsin from red / green cones as shown in Figure 25 (A) or rod rhodopsin as shown in Figure 25 (B) were consistent with this percentage of cone cells. Specific antibodies to rod rhodopsin (rho4D2) were provided by Dr. Robert Molday of University British Columbia and used as previously described (Hicks et al 1986, Exp. Eye Res. 42: 55-71). Rabbit antibodies specific for opsin from red / green cones were purchased from Chemicon (AB5405) and used in accordance with the manufacturer's instructions. Example 10. Intravitreal Administration of Murine Cells in Murine Models for Retinal Degeneration Induced with Oxygen. Newborn wild type C57B16 mice were exposed to hyperoxia (75% oxygen) between postnatal days P7 to P12 is a model of retinal degeneration induced with oxygen (OÍR). Figure 22 illustrates normal postnatal vascular development in C57B16 mice from PO to P30. In PO, only superficial vessels can be seen sprouting around the optic disc. Within a few days, the primary surface network extends to the periphery, reaching the far periphery by the day PlO. Between P7 and P12, the secondary (deep) plexus develops. For P7, a deep and superficial extensive network of vessels is present (FIG 22, insertions). In the following days, a remodeling occurs along with the development of the tertiary (intermediate) layer of vessels until reaching an adult structure approximately in P21. In contrast, in the OIR model, after exposure to 75% oxygen at P7-P12, the normal sequence of events is severely disrupted (FIG 23). The Lin "adult murine HSC populations of the invention were injected intravitreally into P3 in one eye of a mouse that was subsequently subjected to OIR, the other eye was injected with PBS or CD31 negative cells as a control. that the Lin "HSC populations of the present invention can reverse the degenerative effects of high oxygen levels in the developing mouse retina. The fully developed superficial and deep retinal vasculature was observed in P17 in the treated eyes, while the control eyes showed large avascular areas with virtually no deep vessels (FIG 24). Approximately 100 eyes of mice in the OIR model were observed. Normal vascularization was observed in 58% of the eyes treated with the Lin "HSC populations of the invention, compared to 12% of the control eyes treated with CD31 cells" and 3% of the control eyes treated with PBS. RESULTS AND DISCUSSION Murine Retinal Vascular Development; A Model for Ocular Angiogenesis The mouse eye provides a recognized model for the study of retinal vascular development in mammals, such as the vascular development of the human retina. During the development of the murine retinal vasculature, retinal blood vessels directed by ischemia develop in close association with astrocytes. These glial elements migrate over the third trimester of the human fetus, or the neonatal rodent, the retina of the optic disc along the ganglion cell layer and spreads radially. When developing into murine retinal vasculature, endothelial cells use this already established astrocytic template to determine the retinal vascular pattern (see FIG 1 (a and b)). Figure 1 (a and b) represents schematic diagrams of the developing mouse retina. Panel (a) represents the development of the plexus primary (dark lines in the upper left part of the diagram) superimposed on the astrocyte template (light lines), while, (b) represents the second phase of retinal vessel formation. In Figure 1, GCL represents the ganglion cell layer; IPL represents the inner plexus layer; INL represents the inner nuclear layer; OPL remains for the outer plexus layer; ONL represents the outer nuclear layer; RPE represents the epithelium of the retinal pigment; ON represents the optic nerve; and P represents the periphery. At birth, the retinal vasculature is virtually absent. By postnatal day 14 (P14) the retina has developed the primary complex layers (superficial) and secondary (deep) of the retinal vessels coinciding with the onset of vision. Initially, the peripapillary vessels similar to a wheel ray grow radially on the preexisting astrocytic network towards the periphery, becoming progressively interconnected by the formation of the capillary plexus. These vessels grow as a monolayer within the fiber of the nerve through PlO (FIG 1 (a)). Between P7-P8 the collateral branches begin to sprout from this primary plexus and penetrate the retina to the outer plexiform layer where the retinal plexuses, secondary or deep, are formed. In P21, the entire network undergoes remodeling extensive and a tertiary or intermediate plexus is formed on the inner surface of the inner nuclear layer (FIG: 1 (b)). 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, there is a large astrocytic template before the appearance of endogenous blood vessels, allowing an evaluation of the role for cell-cell recognition during a neovascular process. In addition, this consistent and reproducible neonatal retinal vascular process is known to be directed by hypoxia, in this respect it has similarities to many retinal diseases in which ischemia is known to be involved. Enrichment of endothelial progenitor cells (EPC) from bone marrow. Although the expression of the surface cell marker has been extensively evaluated in the EPC population found in the HSC preparations, the markers that uniquely identify EPC are still poorly defined. To enrich the EPC, positive marker cells of hematopoietic lineage (Lin +), i.e., B lymphocytes (CD 45), T lymphocytes (CD3), granulocytes (Ly-6G), monocytes (CD11), and erythrocytes (TER-119), were extracted evacuated from the monouclear cell of the bone marrow of mice. The Sca-1 antigen was used to further enrich the EPC.

A comparison of results obtained after the intravitreal injection of the same number of either Lin cells "Sca-1 + or Lin cells", no difference was detected between the two groups. In fact, when only Lin "Sca-1 ~ cells were injected, a much greater incorporation was observed in the developing blood vessels." Lin "HSC populations of the present invention are enriched with EPCs, based on functional assays. In addition, the Lin + HSC populations behave in a totally different way from the Lin HSC populations, the epitopes commonly used to identify EPC for each fraction (based on previously reported in vitro characterization studies) were also evaluated. these markers were exclusively associated with the Lin fraction ", all increased approximately 70 to approximately 1800% in the Lin" HSC, as compared to the Lin + HSC fraction (FIG.1 (c)) Figure 1 panel (c) illustrates the characterization of the cytometric flow of the separate Lin + HSC and Lin "HSC cells derived from bone marrow. The upper row of the panel (c) shows a dot-diagram distribution of the hematopoietic stem cell of cells not labeled with antibodies. Rl defines the quantifiable interrupted area of positive PE staining. R2 indicates positive GFP. The dot diagrams of Lin "HSC are shown in the Intermediate row and the Lin + HSC dot plots are shown in the lower row. C57B / 6 cells were labeled with PE conjugated antibodies for Sca-1, c-kit, Flk-1 / KDR, CD31. The Tie-2 data were obtained from Tie-2-GFP mice. The percentages at the corners of the dot plots indicate the percentage of cells marked as positive outside the total population of Lin "or Lin + HSC Interestingly, accepted EPC markers similar to Flk-1 / KDR, Tie-2. and Sca-1 were expressed poorly and, therefore, were not used for another fractionation Lin cells "HSC Injected intravitreally contain EPC that recognizes Astrocytes and is incorporated in the Retinal Vasculature in Development. To determine whether the Lin "HSC injected intravitreally can recognize or target specific types of retinal cells, utilizes the astrocytic template and participates in retinal angiogenesis, approximately 10 5 cells of a Lin composition" HSC of the present invention or Lin + HSC cells (control, approximately 105 cells) isolated from the bone marrow of adult mice (transgenic GFP or LacZ) were injected into mouse eyes on postnatal day 2 (P2). Four days after the injection (P6), many cells of the composition Lin "HSC of the present invention, derived from Transgenic GFP or LacZ mice were adherent to the retina and had the characteristic elongated appearance of endothelial cells (FIG 2 (a)). Figure 2 illustrates the engraftment of Lin cells in the retina of the developing mouse As shown in FIG.2, Panel (a), GFPe + Lin + HSC injected intravitreally four days after injection (P6) They join and differentiate in the retina.In many areas of the retinas, the cells expressing GFP were arranged in a pattern that conforms to the implicit astrocytes and the compared blood vessels.These fluorescent cells were observed ahead of the vascular network in endogenous development (FIG.2 (b)) In contrast, only a small number of Lin + HSC (FIG.2 (c)), or adult mouse mesenteric endothelial cells (FIG.2 (d)) was attached to the surface of In order to determine if the cells of a Lin population "injected HSC could also bind to the retinas with already established vessels, we injected a composition Lin" HSC in adult eyes Interestingly, it was not observed that the cells were attached to the Retina or incorporate Rare in normal, established retinal blood vessels (FIG. 2 (e)). This indicates that the Lin "HSC compositions of the present invention do not break a normally developed vasculature and will not initiate abnormal vascularization in the retinas developed normally. To determine the relationship between a Lin "HSC composition of the present invention injected and the retinal astrocytes, a transgenic mouse was used, which expressed a glial fibrillary acidic protein (GFAP, a marker of astrocytes) and a green fluorescent protein directed by a Promoter (GFP): Examination of the retinas of these GFAP-GFP transgenic mice injected with Lin "HSC of transgenic eGFP mice demonstrated the co-localization of GFPe EPC and existing astrocytes (FIG.2 (fh), arrows).

The GFPe + Lin "HSC processes were observed to conform the implicit astrocytic network (arrows, FIG. (g)). The examination of these eyes showed that the labeled, injected cells only linked to astrocytes; in mouse retinas (P6), where the retinal periphery does not yet have endogenous vessels, the injected cells were observed adherent to the astrocytes in these areas are not yet vascularized. Surprisingly, the labeled, injected cells were observed in the deepest layers of the retina at the precise place where the normal retinal vessels will subsequently develop (FIG: 2 (i), arrows). To determine whether the Lin "Injected HSCs of the present invention are stably incorporated into the developing retinal vasculature, the retinal vessels at several time points later. As early as in P9 (seven days after injection), the Lin "HSCs were incorporated into the CD31 + structures (FIG.2 (j)). At P16 (14 days after the injection), the cells were already widely incorporated in vascular-like structures of the retina (FIG.2 (k)). When rhodamine-dextran was injected intravascularly (to identify functional retinal blood vessels) before sacrificing the animals, most of the Lin "HSCs were aligned with the visible vessels. (FIG 2 (1)). Two patterns of distribution of labeled cells were observed: (1) in a standard, the cells were interspersed along the vessels between unlabeled endothelial cells; and (2) the other pattern showed that the vessels were composed entirely of labeled cells. The injected cells were also incorporated into the vessels of the deep vascular plexus (FIG 2 (m)). Although the sporadic incorporation of EPC derived from Lin "HSC into the neovasculature has been previously reported, this is the first report of vascular networks that are totally composed of these cells, which shows that the cells of a population of Lin" HSC derived from marrow Bone of the present invention injected intravitreally can be efficiently incorporated into any layer of the retinal vascular plexus In training. Histological examination of non-retinal tissues (e.g., brain, liver, heart, lung, bone marrow) did not demonstrate the presence of any of the GFP positive cells examined up to 5 or 10 days after the intravitreal injection. This indicates that a sub-population of cells within the Lin "HSC fraction selectively recognizes retinal astrocytes and is stably incorporated in the developing retinal vasculature, because these cells have a lot of endothelial cell characteristics (association with retinal astrocytes). , elongated morphology, stable incorporation in visible vessels and not present in extravascular sites), these cells represent EPC present in the Lin population "HSC. Target astrocytes are the same type observed in many of the hypoxic retinopathies. It is well known that glial cells are a predominant component of the neovascular laminae observed in DR and other forms of retinal injury. Under conditions of reactive gliosis and neovascularization induced with ischemia, the activated astrocytes proliferate, produce cytokines, and up-regulate GFAP, similar to that observed during the formation of the neonatal retinal vascular template in many mammalian species including humans. The Lin "HSC populations of the present invention are will direct or recognize activated astrocytes in adult mouse eyes as they do in neonatal eyes, Lin cells "HSC were injected into adult eyes with retinas lesioned by photocoagulation (FIG: 3 (a)) or needle tip (FIG. (b)) In both models, a population of cells with prominent GFAP staining was observed only around the site of the lesion (FIG: 3 (a and b).) The cells of the Lin "HSC injected compositions located the site of the lesion and remained specifically associated with positive GFAP astrocytes (FIG: 3 (a and b)). At these sites, Lin "HSC cells were also observed to migrate in the deepest layer of the retina at a level similar to that observed during the formation of the deep retinal vasculature.The non-injured portions of the retina did not contain Lin cells" HSC , identical to that observed when Lin "HSC was injected into adult, untreated, normal retinas (FIG 2 (e)). These data indicate that the compositions Lin "HSCs can selectively recognize or target activated glial cells in damaged gliosis adult retinas as well as neonatal retinas that undergo vascularization Lin-HSCs injected intravitreally can Recover and Stabilize Vascular Degeneration. "HSC injected from Intravitreal form recognize astrocytes and are incorporated into the normal retinal vasculature, these cells also stabilize the vasculature that degenerates into ischemic or degenerative retinal diseases associated with gliosis and vascular degeneration. The rd / rd mouse is a model for retinal degeneration that exhibits deep degeneration of the photoreceptor and retinal vascular layers for a month after birth. The retinal vasculature in these mice normally develops until P16 at which time the deeper vascular plexus recedes; in most mice the deep and intermediate plexuses have almost completely degenerated. To determine whether HSC can recover the regressive vessels, Lin + or Lin "HSC (from Balb / c mice) were injected into rd / rd mice into P6. At P33, after injecting Lin + cells, the vessels of the deeper retinal layer were almost totally absent (FIG 4 (a and b).) In contrast, most retinas injected with Lin "HSC in P33 had an almost normal retinal vasculature with three parallel, well-formed, parallel vascular layers (FIG: 4 (a and b)). ). The quantification of this effect showed that the average length of vessels in the deep vascular plexus of rd / rd eyes injected with Lin "was almost three times greater than eyes not treated or treated with Lin + cells (FIG 4 (e)). Surprisingly, the injection of a Lin composition "HSC derived from adult mouse bone marrow rd / rd (FVB / N) also rescued retinal vasculature from neonatal mouse rd / rd that was degenerating (FIG 4 (f)). The degeneration of the vasculature in eyes of mouse rd / rd is observed in as soon as 2-3 postnatal weeks.Injection of Lin "HSC as late as in P15 also resulted in partial stabilization of vasculature in degeneration in rd / rd mice for at least one month (FIG 4 (gyh)). A Lin composition "HSC injected into younger rd / rd mice (eg, P2) was also incorporated into the developing superficial vasculature." At Pll, these cells were observed to migrate to the level of the deep vascular plexus and form a pattern identical to that observed in the outer wild type retinal vascular layer (FIG: 5 (a)). To more clearly describe the manner in which the cells of the Lin "HSC injected compositions are incorporated into, and stabilize the retinal vasculature that degenerates in the rd / rd mice, a Lin composition "HSC derived from Balb / c mice was injected into mouse eyes Tie-2-GFP FVB.FVB mice have the rd / rd genotype and because they express the fusion protein Tie2-GFP All endogenous blood vessels are fluorescent When unlabelled cells of a Lin composition "HSC are injected into neonatal Tie-2-GFP FVB eyes and are Subsequently incorporated into the developing vasculature, there should be no separations in the Tie-2-GFP labeled, endogenous vessels that correspond to the Lin "unlabeled, incorporated HSCs that were injected.The subsequent staining with another vascular marker (eg, CD31) then delineates the entire vessel, allowing determination of whether the non-endogenous endothelial cells are part of the vasculature Two months after the injection, positive CD31 positive, Tie-2-GFP vessels were observed in the retinas of the eyes injected with the Lin composition "HSC (FIG 5 (b)). Interestingly, most of the recovered vessels contained Tie-2-GFP positive cells (FIG 5 (c)). The distribution of pericytes, as determined by smooth muscle actin staining, was not changed by the injection of Lin "HSC, without considering whether there was vascular recovery (FIG 5 * (d)). These data clearly demonstrate that the compositions Lin "HSCs of the present invention injected intravitreally migrate to the retina, participate in the formation of normal retinal blood vessels, and stabilize the endogenous vasculature that is degenerating in a genetically abnormal mouse. Inhibition of Retinal Angiogenesis by Transfected Cells of Lin "HSC, Most Retinal Vascular Diseases they involve abnormal vascular proliferation rather than degeneration. Transgenic cells directed to astrocytes can be used to deliver an anti-angiogenic protein and inhibit angiogenesis. The cells of the Lin "HSC compositions were transfected with T2-tryptophanyl-tRNA synthetase (T2-TrpRS) .T2-TrpRS is a 43 kD fragment of TrpRS that potentially inhibits retinal angiogenesis (FIG 6 (a)) In P12, retinas of eyes injected with a control composition of Lin "HSC transfected with plasmid (without T2-TrpRS gene) in P2 had primary retinal vascular plexuses (FIG. 6 (c)) and secondary (FIG 6 (d)) normal. When the Lin composition "HSC transfected with T2-TrpRS of the present invention was injected into P2 eyes and evaluated 10 days later, the primary network had significant abnormalities (FIG 6 (e)) and the formation of the deep retinal vasculature It was almost completely inhibited (FIG 6 (f)) The few vessels observed in these eyes were markedly attenuated with large gaps between the vessels.The degree of inhibition by Lin "HSCs secreting T2-TrpRS is detailed in Table 2 T2-TrpRS is produced and secreted by cells in the composition of Lin "HSC in vitro and after injection of these transfected cells into the vitreous, a fragment of T2-TrpRs of 30 kD was observed in the retina (FOG. 6 (b)).

This 30 kD fragment was specifically observed only in the retinas injected with the Lin "transfected HSCs of the present invention and this decrease in apparent molecular weight compared to the recombinant protein or synthesized in vitro may be due to the process or degradation of T2 -TrpRS in vivo These data indicate that Lin "HSC compositions can be used to deliver functionally active genes, such as genes expressing angiostatic molecules, to the retinal vasculature by targeting or recognizing activated astrocytes. Although it is possible that the observed angiostatic effect is due to cell-mediated activity, this is very unlikely since eyes treated with Lin compositions "HSC identical, but not transfected with T2, had normal retinal vasculature." Table 2. Inhibition Vascular by the Lin "HSCs that secrete T2-TrpRS Primary plexus Deep plexus Inhibited Normal Complete Partial Normal T2-TrpRS 60% 40% Jo. "6 60% 6.7% (15 eyes) (9 eyes) (6 eyes) (5 eyes) (9 eyes) (1 eye) Control 0% 100% 0% 38.5% 61.5% (13 eyes) (0 eyes) ( 13 eyes) (0 eyes) (5 eyes) (8 eyes) The Lin "HSC populations injected intravitreally localize the retinal astrosites, are incorporated into the vessels, and may be useful in the treatment of many retinal diseases. Although most cells of injected HSC compositions adhere to the astrocytic template, a small number migrate deep into the retina, limiting the regions where the deep vascular network will subsequently develop. Although none of the positive GFAP astrocytes were observed in this area before 42 days postnatally, this does not exclude the possibility that GFAP-negative glial cells are already present to provide a signal for the localization of Lin "HSC. that in many diseases are associated with reactive gliosis, in DR, in particular, glial cells and their extracellular matrix are associated with pathological angiogenesis, because the cells of the Lin "HSC injected compositions bind specifically to the glial cells that express GFAP Without considering the type of lesion, the Lin "HSC compositions of the present invention can be used to recognize pre-angiogenic lesions in the retina For example, in ischemic retinopathies such as diabetes, neovascularization is a hypoxia response. By targeting the compositions of Lin ~ HSC to sites of pathological neovascularization, the neova developing sculature can be stabilized by preventing neovasculature abnormalities such as hemorrhage or edema (causes of vision loss associated with DR) and can potentially alleviate the hypoxia that originally stimulated neovascularization. Abnormal blood vessels can be restored to normal condition. Additionally, angiostatic proteins, such as T2-TrpRS can be delivered to pathological angiogenesis sites using Lin compositions "transfected HSC and laser-induced astrocyte activation.Since laser photocoagulation is commonly used in clinical ophthalmology, this approach has Applications for many retinal diseases Although such cell-based approaches have been explored in cancer therapy, their use for eye diseases is more convenient since intraocular injection makes it possible to deliver large numbers of cells directly to the site of the disease Neurotrophic and Vascul Root Recovery by Lin "HSC. MACS was used to separate Lin "HSC from the bone marrow of C3H (rd / rd), FVB (rd / rd) mice of enhanced green fluorescent protein as described above.The Lin" HSC containing EPC from these mice were injected from Intravitreal manner in mouse eyes P6 C3H or FVB. The retinas were collected at various time points (1 month, 2 months and 6 months) after the injection. The vasculature was analyzed by confocal microscope examination laser after staining with CD31 antibodies and retinal histology after nuclear staining with DAPI. The analysis of microarray gene expression of retinal mRNA at variable time points was also used to identify genes potentially involved in the effect. The eyes of rd / rd mice had profound degeneration of both neurosensory retina and retinal vasculature to P21. The eyes of rd / rd mice treated with Lin "HSC in P6 maintained a normal retinal vasculature over six months, both intermediate and deep layers were significantly improved when compared with the controls at all time points (1M, 2M , and 6M) (see Fig. 12) In addition, we observed that the retinas treated with Lin "HSC were also thicker (1MM 1-2 times, 2M, 1-3 times, 6M, 1-4 times) and had higher number of cells in the outer nuclear layer (IM; 2.2 times, 2M; 3.7 times, 6M; 5.7 times) with respect to eyes treated with Lin "HSC as a control." Large-scale genomic analysis of "recovered" (eg, Lin "HSC) compared to the control rd / rd retinas (untreated or not treated with Lin ") demonstrated a significant over-regulation of genes encoding sHSPs (small heat shock proteins) and specific growth factors that are correlated with the vascular and neural recovery, including genes encoding the proteins listed in FIG. 20, panels A and B. Lin populations "HSCs derived from the bone marrow of the present invention reproducibly and significantly induced maintenance of a normal vasculature and dramatically increased the photoreceptor and other neuronal cell layers in the rd / rd mouse. Neurotrophic recovery effect was correlated with significant over-regulation of small heat shock proteins and developing factors and provides insights into the therapeutic approaches for currently undetectable retinal degenerative disorders.Redl Rdl / Rdl Retinas Exhibit Deep Vascular and Neuronal Degeneration The normal postnatal retinal vascular and neuronal development in mice has been well described and is analogous to the changes observed in the third trimester of the human fetus (Dorrell et al., 2002, Invest. Ophthalmol, Vis. Sci. 43: 3500-3510). Homozygous mice for the rdl gene share many characteristics of human retinal degeneration (Frasson et al., 1999, Nat. Med. 5: 1183-1187) and exhibit a rapid photoreceptor (PR) loss accompanied by severe vascular atrophy as the result of a mutation in the gene encoding phosphodiesterase PR cGMP (Bowes et al., 1990, Nature 347: 677-680). To examine the vasculature during retinal development and its subsequent degeneration, antibodies were used against collagen IV (CIV), an extracellular matrix protein (ECM) of mature vasculature, and CD31 (PECAM-1), a marker for endothelial cells. (FIG.15). The retinas of rdl / rdl (C3H / HeJ) developed normally until approximately the postnatal day (P) 8 when the degeneration of the outer nuclear layer containing the photoreceptor (ONL) began. The ONL degenerated rapidly and the cells died by apoptosis such that only a single layer of the nucleus remained at P20. Double staining of completely mounted retinas with antibodies for both CIV and CD31 revealed details of vascular degeneration in rdl / rdl mice similar to that described by others (Blanks et al., 1986, J. Comp.Neurol. 254: 543- 553). The primary and deep retinal vascular layers appeared to develop normally at P12 after which there is a rapid loss of endothelial cells as evidenced by the absence of CD31 staining. CD31 positive endothelial cells were present in a normal distribution through P12 but disappeared rapidly after that. Interestingly, positive CIV staining remained present throughout all the time points examined, suggesting that the vessels and associated ECM were formed normally, but only the matrix remained after the P13 time in which no CD31 positive cells were observed. (FIG 15, intermediate panels). The intermediate vascular plexus also degenerated after P21, but the evolution is slower than that observed in the deep plexus (FIG 15, upper panel). The neural and vascular retinal cell layers of a normal mouse are shown by comparison for the rdl / rdl mouse (right panels, FIG.15). The Neuroprotective Effect of the Lin "HSCs Derived from the Bone Marrow in rdl / rdl Mice The Lin" Intravitreally injected HSCs are incorporated into the endogenous retinal vasculature in all three vascular plexuses and prevent the degeneration of the vessels. Interestingly, the injected cells are never virtually observed in the outer nuclear layer. These cells are incorporated into the retinal vessels under formation or are observed in close proximity to these vessels. The Lin "murine HSCs (from C3H / HeJ) were injected intravitreally in mouse eyes (rdl / rdl) C3H / HeJ in P6, just before the onset of degeneration.At P30, the eyes injected with control cell (CD31 ~) showed the typical rdl / rdl phenotype, ie, the almost complete degeneration of the deep vascular plexus and the ONL was observed in each retina examined.The eyes injected with the Lin ~ HSCs maintained intermediate and deep vascular plexuses that appeared normal. Surprisingly, significantly more cells were observed in the internuclear layer (INL) and the ONL of eyes injected with Lin "HSC than in the eyes injected with control cells (FIG.16 (A)) .This effect of recovery of the Lin" HSCs it could be observed at 2 months (FIG 16 (B)) and for 6 months after the injection (FIG 16 (C)). Differences in the vasculature of the intermediate and deep plexus of eyes injected with Lin "HSC, as well as the INL and ONL containing neuronal cells, were significant at all time points measured when the recovered and non-recovered eyes were compared (FIG. 16 (B and C)) This effect was quantified by measuring the total length of the vasculature (FIG 16 (D)) and counting the number of positive DAPI cell nuclei observed in the ONL (FIG 16 (E)). Simple linear regression analysis was applied to the data at all time points.A statistically significant correlation was observed between vascular recovery and neuronal recovery (eg, ONL thickness) at P30 (p <0.024) and P60 (p <0.034) in the eyes injected with Lin "HSC (FIG 16 (F)). The correlation remained high, although not statistically significant (p <0.014) in P18 when the retinas injected with Lin "HSC were compared with the injected retinas with control cells (FIG, 16 (F)). In contrast, the retinas injected with control cells did not show any significant correlation between the preservation of the vasculature and the ONL, at any time point (FIG 16 (F)). These data demonstrated that intravitreal injection of Lin "HSCs results in concomitant retinal vascular and neuronal retinal recovery in mouse rdl / rdl retinas No cells injected into the ONL or anywhere other than within, or in close proximity to, the vessels were observed Retinal blood tests Functional Recovery of rd / rd Retinas injected with Lin ~ HSC Electroretinograms (ERGs) were carried out in mice 2 months after the injection of control cells or Lin "murine HSC (FIG.17). Microscopic and immunohistochemical analysis was performed with each eye following the ERG records to confirm that vascular and neuronal recovery had occurred. The representative ERG records of unrecovered, treated, recovered and controlled eyes show that in the recovered eyes, the digitally subtracted signal (treated eyes less untreated) produced a clearly noticeable signal with an amplitude in the order of 8-10 microvolts ( FIG 17). Clearly, the signals from both eyes are severely abnormal. However, consistent and perceptible ERGs were recordable from eyes treated with Lin "HSC." In all cases, the ERG of the eye control was not noticeable. Although the amplitudes of the signals in the recovered eyes were considerably lower than the normal ones, the signals were consistently observed as long as there was histological recovery and they were in the order of magnitude of those reported by other recovery studies, based on genes. All these results are the sample of some degree of functional recovery in the eyes treated with the Lin "HSCs of the invention." The rd / rd recovered types of recovered cells are predominantly cones.The recovered and unrecovered retinas were analyzed immunohistochemically with specific antibodies. for rod or cone opsina The same eyes used for the ERG records presented in FIG.17 were analyzed for rods or cones opsina.In wild type mouse retinas, less than about 5% of the photoreceptors present are cones (FIG. Soucy et al., 1998, Neuron 21: 481-493) and the immunohistochemical staining patterns observed with opsin of red / green cones as shown in FIG. 25 (A) or rod rhodopsin as shown in FIG. (B), were consistent with this percentage of cone cells.When the wild-type retinas were stained with pre-immune IgG, no staining was observed anywhere in the cells. neurosensory retinas different from auto-fluorescence of the blood vessels (FIG 25 (C)). Two months after birth, the retinas of the rd / rd non-injected mice had an essentially atrophic outer nuclear layer that did not show any staining with red / green cones opsin (FIG. 25. (D)) or rhodopsin (FIG. 25 (G)). The injected eyes with control, CD31-HSC also did not stain positively for the presence of any cone opsin (FIG 25 (E)) or cane (FIG, 25 (H)). In contrast, contralateral eyes injected with Lin "HSC had approximately 3 to about 8 rows of nuclei in a conserved outer nuclear layer, most of these cells were positive for cone opsin (FIG. 25 (F)) with approximately 1- 3% positive for cane opsin (FIG.25 (I)) .Remarkably, this is almost the inverse of what is ordinarily observed in the normal mouse retina, which is dominated by rods. Lin "HSC preserves the cones for prolonged periods of time during which they would degenerate ordinarily. The Lin "HSCs Derived from the Human Bone Marrow (hBM) also Recover Retinas in Degeneration The Lin" HSCs isolated from the human bone marrow behave similarly to the Lin "murine HSCs.The bone marrow was harvested from human donors and the Lin + HSCs were evacuated, producing a population of Lin "human HSCs (hLin" HSCs). These cells were labeled ex vivo with fluorescent ink and injected into mouse eyes C3SnSmn.CB17-Pr.irdc SCID. The Lin "Injected HSCs migrated to and recognized sites of retinal angiogenesis in a manner identical to that seen when Lin" murine HSCs were injected (FIG.18 (A)). In addition to the vascular recognition, the Lin "human HSCs also provided a strong recovery effect in both the vascular and neuronal cell layers of the rdl / rdl mice (FIG.18 (B and C).) This observation confirms the presence of cells in the human bone marrow that recognize retinal vasculature and can prevent retinal degeneration The Lin "HSCs have Vasculo- and Neurotrophic Effects in the rdlO / rdlO mouse. Although the rdl / rdl mouse is the most widely used model and the best characterized for retinal degeneration (Chang et al, 2002, Vision Res. 42: 517-525), the degeneracy is very rapid, this aspect differs from the time course more slow, usual, observed in human disease. In this strain, the cellular degeneration of the photoreceptor begins around P8, at which time the retinal vasculature is still expanding rapidly (FIG 15). The subsequent degeneration of the deep retinal vasculature occurs even when the intermediate plexus is still forming and, thus, the retinas of the rdl / rdl mice never fully develop, unlike what is observed in most humans with this disease. A rdl O mouse model, which has a slower time course of degeneration and more closely resembles the human retinal degenerative condition, was used to investigate vascular recovery mediated by Lin ~ HSC. In the rdl O mouse, the photoreceptor cell degeneration begins around P21 and vascular degeneration begins shortly afterwards. Because the normal neurosensory retinal development is very complete by P21, it was observed that the degeneration begins after the retina has completed differentiation and thus is more analogous to human retinal degeneration than the rdl / rdl mouse model. Control cells or Lin "HSCs from rd20 mice were injected into P6 eyes and retinas were evaluated at variable time points.At P21 the retinas of both eyes injected with control cells and Lin" HSC appeared normal with full development of all vascular layers and normal development of INL and ONL (FIG.18 (D and H)). At approximately P21 retinal degeneration began and evolved with age. At P30, retinas injected with control cells showed severe vascular and neuronal degeneration (FIG. 18 (1)), whereas retinas injected with Lin "HSC maintained the vascular layers and the photoreceptor cells almost normal (FIG.18 (E)) .The difference between the recovered and unrecovered eyes was more pronounced at later time points (compare FIG. and G) with 18 (J and K).) In eyes treated with control, the evolution of vascular degeneration was observed very clearly by immunohistochemical staining with CD31 and collagen IV (FIG.18 (IK)). control were almost completely negative for CD31, while vascular "fingerprints" positive for collagen IV remained evident, indicating that vascular regression more than incomplete vascular formation had occurred.In contrast, eyes treated with Lin "HSC had both positive vessels of CD31 and collagen IV that looked very similar to normal wild type eyes (compare FIG 18 (F and I)). Retinal Mouse Expression Analysis rd / rd after treatment with Lin HSC A large-scale genomic system (microarray analysis) was used to analyze recovered and unrecovered retinas to identify putative mediators of neurotrophic recovery. of genes in rdl / rdl mouse retinas treated with Lin "HSCs was compared with non-injected retinas as well as with retinas injected with control cells (CD31 ~). These comparisons were carried out each one in triplicate. To be considered as present, the genes were required to have expression levels at least 2 times higher than the basic levels in all three triplicates. Genes that were up-regulated 3-fold in the retinas protected with Lin "HSC compared to the rd / rd mouse retinas not injected and injected with control cells are shown in FIG 20, panels A and B. The coefficient of the variance levels (VOC) were calculated for the expressed genes by dividing the standard deviation by the average expression level of each cRNA replica, and the correlation between the expression levels and the noise variance was calculated correlating the standard deviation (SD) ) and average, a correlation was obtained between the level of expression of the gene and the standard deviation for each gene, allowing the basic levels and thresholds of the level of reliable expression to be determined.Fullly, the data are well within the acceptable limits (Tu et al., 2002, Proc. Nati, Acad. Sci. USA 99: 14031-14036) The genes described individually, later, exhibited expression levels by above these critical expression levels. The "t-test" values in pairs for the described genes are also presented in Table 1. In each case, the p-values are reasonable (close to or less than 0. 05), which shows that there are similarities between the replicates and the probable significant differences between the different test groups. The majority of significantly overregulated genes, including MAD and Ying Yang-1 (YY-1) (Austen et al., 1997, Curr. Top, Microbiol.Immunol., 224: 123-130) encode proteins with functions that involve protection of apoptosis cells. A number of crystal genes, which have sequence homology and functions similar to known heat shock proteins involving the protection of tension cells, were also over-regulated by treatment with Lin "HSC. α-crystalline was localized to the ONL by immunohistochemical analysis (Figure 19) Figure 19 shows that the aa crystalline is over-regulated in the outer nuclear layer cells recovered after treatment with Lin " HSC but not in contralateral eyes treated with control cells. The left panel shows IgG staining (control) in retina recovered. The intermediate panel shows aa crystalline in a recovered retina. The right panel shows aa crystalline in an unrecovered retina. The rdl / rdl mouse retinal messenger RNA retrieved with Lin "human HSCs were hybridized to human specific Affymetrix U133A microarray chips.After strict analysis, a number of genes whose mRNA expression was specific to humans, above basic, and significantly higher in retinas recovered with Lin "human HSC compared to retinas recovered with Lin" murine HSC and unrecovered retinas injected with human control cells (FIG 20 panel C). CD6, a cell adhesion molecule expressed on the surface of primitive and recently differentiated CD34 + hematopoietic stem cells, and interferon alfa 13, another gene expressed by hematopoietic stem cells, were both found using the microarray bioinformatics technique, validating the evaluation protocol . In addition, several growth factors and neurotrophic factors were expressed above background using mouse retina samples recovered by Lin "human HSC (FIG: 20, panel D.) Markers for consigned lineage hematopoietic cells were used to select negatively a population of Lin ~ HSC derived from the bone marrow containing EPC.Although the sub-population of Lin ~ HSC derived from bone marrow that can serve as EPC is not characterized by commonly used cell surface markers, the behavior of these cells in the injured or developing retinal vasculature is completely different than that observed for populations adult endothelial cells or Lin +. These cells selectively recognize sites of retinal angiogenesis and participate in the formation of visible blood vessels. Hereditary retinal degenerative diseases are often accompanied by loss of retinal vasculature. Effective treatment of such diseases requires restoration of function as well as maintenance of complex tissue architecture. Although several recent studies have explored the use of cell-based administration of trophic factors or stem cells alone, some combination of both may be necessary. For example, the use of growth factor therapy to treat retinal degenerative disease resulted in irregular overgrowth of blood vessels resulting in a severe alteration of the normal retinal tissue architecture. The use of retinal or neural stem cells to treat a retinal degenerative disease can reconstitute neuronal function, but a functional vasculature will also be necessary to maintain the functional integrity of the retina. The incorporation of Lin ~ HSCs cells of the present invention into the retinal vessels of rd / rd mice stabilized the degenerative vasculature without altering the retinal structure. This recovery effect was also observed when cells were injected into rd / rd mice in P15. Because vascular degeneration begins at P16 in rd / rd mice, this observation extends the therapeutic window for effective treatment with Lin "HSC.Retinal neurons and photoreceptors are preserved and visual function is maintained in eyes injected with the Lin. "HSC of the present invention. Lin ~ HSCs derived from the adult bone marrow exert profound vasculo- and neurotrophic effects when injected intravitreally in mice with retinal degenerative disease. This recovery effect persists for up to 6 months after treatment and is most effective when Lin "HSCs are injected prior to complete degeneration of the retina (up to 16 days after birth in mice that ordinarily show complete retinal degeneration at 30 days postnatal) This recovery is observed in 2 mouse models of retinal degeneration and, notably, can be performed with HSCs derived from human adult bone marrow when the recipient is an immunodeficient rodent with retinal degeneration (eg, the SCID mouse) or when The donor is a mouse with retinal degeneration, although several recent reports have described a partial phenotypic recovery in mice or dogs with retinal degeneration after genetic base genetic recovery with the gene. wild type (Ali, et al, 2000. Nat. Genet .25: 306-310; Takahashi et al., 1999; J. Virol. 73: 7812-7816; Acland et al., 2001. Nat. Genet. 28: 92- 95.), the present invention is the first therapeutic effect based on generic cells achieved by vascular recovery. Therefore, the potential utility of such an approach in the treatment of a group of diseases (e.g., retinitis pigmentosa) with more than 100 known associated mutations is more practical than creating individual genetic therapies to treat each known mutation. The precise molecular basis of the neurotrophic recovery effect remains unknown, but is only observed when there is a concomitant vascular recovery / stabilization. The presence of injected stem cells, per se, is not sufficient to generate a neurotrophic recovery and the clear absence of neurons derived from stem cells in the outer nuclear layer excludes the possibility that the injected cells are transformed into photoreceptors. The data obtained by analyzing microarray gene expression demonstrate significant over-regulation of genes known to have anti-apoptotic effects. Because much of the neuronal death observed in retinal degeneration is due to apoptosis, such protection can be of great therapeutic benefit in prolonging life of photoreceptors and other critical neurons for visual function in these diseases. C-myc is a transcription factor that participates in apoptosis by over-regulation of the various factors that induce downstream apoptosis. The expression C-myc was increased 4.5 times in rd / rd mice with respect to the wild type indicating a potential complication in the photoreceptor degeneration observed in the rdl / rdl mouse. Mad 1 and YY-1, two genes dramatically over-regulated in retinas protected with Lin "HSC (Fig. 20, panel A), are known to suppress the activity of c-myc, thereby inhibiting the apoptosis induced by c-myc. The over-expression of Madl has also been shown to suppress the Fas-induced activation of caspase 8, another critical component of the apoptotic paths.The over-regulation of these two molecules may have a role in protecting the retina from neural degeneration and vascular preventing the initiation of apoptosis that normally leads to degeneration in rd / rd mice Another group of genes that were extensively over-regulated in retinas protected with Lin "HSC includes members of the crystalline family (Fig. 20, panel B ). Similar to heat shock proteins and other stress-induced proteins, crystallins can be activated by retinal tension and provide a protective effect against apoptosis. The abnormally low expression of aA-crystalline is correlated with the loss of the photoreceptor in a rat model of retinal dystrophy and a recent proteomic analysis of the retina in the mouse rd / rd showed the induction of the over-regulation of the crystalline in response to retinal degeneration. Based on our microarray data of rd / rd mouse retinas recovered with EPC, the up-regulation of crystallins seem to have a key role in retinal neuroprotection mediated by EPC. Genes such as c-myc, Madl, Yx-1 and crystallins are probably mediators downstream of neuronal recovery. Neurotrophic agents can regulate apoptotic gene expression, although our microarray analysis of retinas recovered with mouse stem cells showed no induction of increased levels of known neurotrophic factors. The analysis of the recovery mediated by stem cells derived from human bone marrow with specific human chips, on the other hand, showed low, but significant increases in the expression of multiple growth factor genes. The over-regulated genes include several members of the fibroblast growth factor family and otoferlina. Mutations in the otoferlina gene are associated with genetic disorders that lead to deafness due to auditory neuropathy. It is possible that the production of otoferlina by Lin "HSCs injected contributes to the prevention of retinal neuropathy as well." Historically, it has been assumed that the vascular changes observed in patients and animals with retinal degeneration were secondary to decreased metabolic demand at the time of photoreceptor death. The present data indicate that, at least for mice with hereditary retinal degeneration, preserving the normal vasculature may help maintain the components of the outer nuclear layer as well.Recent reports in the literature would support the concept that tissue-specific vasculature has effects trophies that go beyond what is expected to simply provide vascular "reconstruction." For example, endothelial cells of the liver can be induced to produce, after VEGFR1 activation, critical growth factors for hepatocyte regeneration and maintenance on the face of the liver injury ica (LeCouter et al., 2003, Science 299: 890-893). Similar indicative interactions between vascular endothelial cells and adjacent hepatic parenchymal cells are reportedly involved in the organogenesis of the liver, or before the formation of functional blood vessels. The endogenous retinal vasculature in individuals with retinal degeneration may not facilitate such a dramatic recovery, but if this vasculature is supported with endothelial progenitors derived from hematopoietic stem cell populations of bone marrow, they can make the vasculature more resistant to degeneration and at the same time facilitate retinal as well as vascular neuronal survival. In humans with retinal degeneration, delaying the onset of complete retinal degeneration can provide years of additional vision. The animals treated with the Lin "HSCs of the present invention had significant preservation of an ERG, which may be sufficient to maintain vision Clinically, it is widely appreciated that there may be a substantial loss of photoreceptors and other neurons while preserving the Functional vision At some point, the critical threshold is crossed and vision is lost.As almost all human hereditary retinal degenerations are of early onset, but slow, an individual with retinal degeneration can be identified and treated intravitreally with an autologous bone marrow stem cell graft of the invention to delay retinal degeneration and concomitant loss of vision To increase the recognition and incorporation of the stem cells of the invention, the presence of activated astrocytes is desirable (Otani et al. 2002, Nat.

Med. 8: 1004-1010); this can be brought to capo by early treatment when there is an associated gliosis, or by using a laser to stimulate the local proliferation of the activated astrocytes. Optionally, ex vivo transfection of the stem cells with one or more neurotrophic substances before the intraocular injection can be used to increase the recovery effect. This approach can be applied to the treatment of other visual neuronal degenerative disorders, such as glaucoma, in which there is a cellular degeneration of the retinal ganglion. The "HSC" Lin populations of the present invention contain a population of EPC that can promote angiogenesis by recognizing reactive astrocytes and being incorporated into an established template without altering the retinal structure The Lin ~ HSC of the present invention also provides a surprising recovery effect Long-term neurotrophic in eyes suffering from retinal degeneration In addition, Lin's compositions of "autologous, genetically modified • HSC containing EPC can be transplanted into ischemic or abnormally vascularized eyes and can be stably incorporated into new vessels and neuronal layers and administered continuously therapeutic molecules locally for extended periods of time. Such local administration of genes that express pharmacological agents in physiologically significant doses represents a new paradigm for the treatment of ocular diseases not currently treatable. The photoreceptors in the normal mouse retina, for example, are predominantly rods, but the outer nuclear layer observed after recovery with the Lin ~ HSCs of the invention contained predominantly cones. Most hereditary human retinal degenerations occur as a result of specific defects in primary rods, and the loss of cones is secondary to rod dysfunction, which is probably related to the loss of some trophic factor expressed by rods. The present method to induce cone survival despite the retinal degeneration / rods facilitated by Lin "HSC, provides a way to better preserve the conical dominated human macula in diseases such as retinitis pigmentosa." Numerous variations and modifications of the modalities described above may be effected without departing from the spirit and scope of the novel aspects of the invention.No limitation with respect to the specific embodiments illustrated herein is intended or should be inferred.

Claims (49)

1. NOVELTY OF THE INVENTION Having described the invention as above, the content is claimed as contained in the following: CLAIMS 1. A method for alleviating cellular degeneration of cones in the retina of a mammal, characterized in that it comprises the step of administering to the retina of a mammal suffering from an eye disease a population of isolated, negative lineage hematopoietic stem cells derived from the bone marrow of a mammal, comprising hematopoietic stem cells and endothelial progenitor cells, in an amount sufficient to delay the cellular degeneration of cones in the retina.
2. 2. The method according to claim 1, characterized in that at least about 20% of the cells in the population of hematopoietic stem cells, of negative lineage, isolated, express the CD31 surface antigen.
3. 3. The method according to claim 1, characterized in that at least about 50% of the cells in the hemopoietic stem cell population, isolated negative lineage, express the CD31 surface antigen.
4. 4. The method according to claim 1, characterized in that at least about 75% of the cells in the population of hematopoietic stem cells, of negative lineage, isolated, express the CD31 surface antigen.
5. The method according to claim 1, characterized in that at least about 50% of the cells in the population of hematopoietic stem cells, of negative lineage, isolated, express the surface antigen for integrin a6.
6. 6. The method according to claim 1, characterized in that the isolated, negative lineage hematopoietic stem cell population is obtained from adult bone marrow.
7. The method according to claim 1, characterized in that the population of hematopoietic stem cells, of negative lineage, isolated comprises murine cells.
8. The method according to claim 7, characterized in that at least about 50% of the cells in the population of hematopoietic stem cells, of negative lineage, isolated, express the surface antigen CD31 and at least about 50% of the cells express the CD117 surface antigen.
9. 9. The method according to claim 7, characterized in that at least about 65% of the cells in the stem cell population hematopoietic, negative lineage, isolated, express the CD117 surface antigen.
10. The method according to claim 7, characterized in that at least about 80% of the cells in the population of hematopoietic stem cells, of negative lineage, isolated, express the surface antigen CD31 and at least about 70% of the cells express the CD117 surface antigen.
11. 11. The method according to claim 1, characterized in that the population of hematopoietic stem cells, of negative lineage, isolated, comprises human cells.
12. The method according to claim 11, characterized in that the cells in the hemopoietic stem cell population, isolated negative lineage, are negative for CD133, at least about 50% of the cells express the surface antigen for integrin a6 , and at least about 50% of the cells express the CD31 surface antigen.
13. The method according to claim 11, characterized in that the cells in the hemopoietic stem cell population, isolated negative lineage, are positive for CD133, less than about 30% of the cells express the surface antigen for a6 integrin, and less than about 30% of the cells express the CD31 surface antigen.
14. The method according to claim 1, characterized in that it includes the additional step of isolating the population of hematopoietic stem cells from the mammal suffering from the ocular disease before administering the cells to the retina.
15. The method according to claim 14, characterized in that the population of hematopoietic stem cells, of negative lineage, is isolated by: (a) extracting bone marrow from the mammal to be treated; (b) separating a plurality of monocytes from the bone marrow; (c) label the monocytes with a panel of lineage antibodies conjugated with biotin to one or more surface lineage antigens selected from the group consisting of CD2, CD3, CD4, CD11, CDlla, Mac-1, CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, Ly-6G, TER-119, CD45RA, CD56, CD64, CD68, CD86, CD66b, HLA-DR, and CD235a; Y (d) removing monocytes that are positive for the one or more lineage surface antigens from the plurality of monocytes and recovering a population of negative lineage hematopoietic stem cells containing endothelial progenitor cells.
16. 16. The method according to claim 15, characterized in that the mammal is a mouse.
17. 17. The method according to claim 15, characterized in that the mammal is a mouse and the monocytes are marked in step (c) with a panel of lineage antibodies conjugated with biotin to CD3, CD11, CD45, Ly-6G, and TER -119.
18. 18. The method according to claim 15, characterized in that the mammal is a human.
19. 19. The method according to claim 15, characterized in that the mammal is a human and the monocytes are labeled in step (c) with a panel of lineage antibodies conjugated with biotin to CD2, CD3, CD4, CDlla, Mac- 1, CD14, CD16, CD19, CD33, CD38, CD45RA, CD64, CD68, CD86, and CD235a.
20. The method according to claim 18, characterized in that the mammal is a human and the method includes the additional steps of labeling the monocytes with a CD133 antibody conjugated with biotin and recovering a population of hematopoietic stem cells of negative lineage, positive for CD133.
21. The method according to claim 18, characterized in that the mammal is a human and the method includes the additional steps of labeling the monocytes with a CD133 antibody conjugated with biotin, removing the CD133-positive cells, and recovering a population of cells hematopoietic stem of lineage negative, negative for CD133.
22. 22. The method according to claim 1, characterized in that the population of hematopoietic stem cells, negative lineage, isolated is administered by intraocular injection.
23. 23. The method according to claim 22, characterized in that the disease is a retinal degenerative disease.
24. 24. The method according to claim 22, characterized in that the disease is an ischemic retinopathy.
25. 25. The method according to claim 22, characterized in that the disease is a vascular hemorrhage.
26. 26. The method of compliance with the claim 22, characterized in that the disease is a vascular leak.
27. 27. The method of compliance with the claim 22, characterized in that the disease is a choroidopathy.
28. 28. The method of compliance with the claim 22, characterized in that the disease is a macular degeneration related to age.
29. 29. The method of compliance with the claim 22, characterized in that the disease is diabetic retinopathy.
30. 30. The method according to claim 22, characterized in that the disease is an alleged ocular histoplasmosis.
31. 31. The method according to claim 22, characterized in that the mammal is a neonate mammal.
32. 32. The method according to claim 31, characterized in that the disease is retinopathy of prematurity.
33. 33. The method according to claim 22, characterized in that the disease is a sickle-cell anemia.
34. 34. The method according to claim 22, characterized in that the disease is retinitis pigmentosa.
35. 35. The method according to claim 1, characterized in that the population of isolated negative lineage hematopoietic stem cells is transfected with a gene that functionally encodes a therapeutically useful peptide before administering the stem cells to the mammalian retina.
36. 36. The method according to claim 35, characterized in that the therapeutically useful peptide is an anti-angiogenic peptide.
37. 37. The method according to the claim 35, characterized in that the anti-angiogenic peptide is a protein fragment.
38. 38. The method of compliance with the claim 37, characterized in that the protein fragment is an anti-angiogenic fragment of TrpRS.
39. 39. The method of compliance with the claim 38, characterized in that the TrpRS fragment is T2-TrpRS.
40. 40. The method according to claim 35, characterized in that the therapeutically useful peptide is a neurotrophic agent.
41. 41. The method according to claim 40, characterized in that the neurotrophic agent is selected from the group consisting of nerve growth factor, neurotrophin-3, neurotrophin-4, neurotrophin-5, ciliary neurotrophic factor, derived neurotrophic factor of the pigmented retinal epithelium, insulin-like growth factor, neurotrophic factor derived from the glial cell line, and neurotrophic factor derived from the brain.
42. 42. The method according to claim 35, characterized in that the population of hematopoietic stem cells, of negative, transfected lineage, is prepared by: (a) extracting bone marrow from an adult mammal; (b) separating a plurality of monocytes from the marrow that is; (c) labeling the plurality of monocytes with a panel of lineage antibodies conjugated with biotin to CD2, CD3, CD4, CD11, CDlla, Mac-1, CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, Ly -6G, TER-119, CD45RA, CD56, CD64, CD68, CD86, CD66b, HLA-DR, and CD235a; and (d) separating monocytes that are positive for one or more lineage surface antigens from the plurality of monocytes and recovering a population of negative lineage hematopoietic stem cells containing endothelial progenitor cells; and (e) transfecting negative lineage hematopoietic stem cells recovered in step (d) with a polynucleotide functionally encoding a therapeutically useful peptide.
43. 43. A method of preserving cone cells in the retina of a mammal suffering from and an ocular disease, characterized in that it comprises isolating from the bone marrow of the mammal a population of hematopoietic stem cells of negative lineage including endothelial progenitor cells and subsequently Intravitreally injecting the isolated stem cells into an eye of the mammal in a sufficient number to alleviate the degeneration of cone cells in the retina.
44. 44. The method of compliance with the claim 43, characterized in that the number of stem cells is effective to repair retinal damage of a mammalian eye.
45. 45. The method according to claim 43, characterized in that the number of stem cells is effective to stabilize the retinal neovasculature of the eye of the mammal.
46. 46. The method according to claim 43, characterized in that the number of stem cells is effective to mature the retinal neovasculature of the eye of the mammal.
47. 47. The method according to claim 43, characterized in that the disease is a retinal degenerative disease.
48. 48. The method according to claim 43, characterized in that the population of isolated negative lineage hematopoietic stem cells is transfected with a gene that functionally encodes a therapeutically useful peptide before administering the stem cells to the retina of the mammal.
49. 49. A method of preserving cone cells in the retina of a mammal suffering from and an ocular disease, characterized in that it comprises isolating from the bone marrow of a mammal a population of hematopoietic stem cells of negative lineage including cells Endothelial progenitors, treat the retina with a laser to stimulate the local proliferation of activated astrocytes in the retina, and subsequently intravitreally inject the isolated stem cells into the mammalian eye in a sufficient number to alleviate cone cell degeneration in the retina .