Hematopoietic stem cells and methods of treatment of neovascular eye diseases therewith

Abstract

Isolated, mammalian, adult bone marrow-derived, lineage negative hematopoietic stem cell populations (Lin − HSCs) contain endothelial progenitor cells (EPCs) capable of rescuing retinal blood vessels and neuronal networks in the eye. Preferably at least about 20% of the cells in the isolated Lin − HSCs express the cell surface antigen CD 31 . The isolated Lin − HSC populations are useful for treatment of ocular vascular diseases. 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 − HSC population containing EPCs. Isolated Lin − HSCs that have been transfected with therapeutically useful genes are also provided, and are useful for delivering genes to the eye for cell-based gene therapy. Methods of preparing isolated stem cell populations of the invention, and methods of treating ocular diseases and injury are also described.

1 . A method of isolating an adult bone marrow-derived, lineage negative hematopoietic stem population including endothelial progenitor cells comprising the steps of:
 (a) extracting bone marrow from an adult mammal;   (b) separating a plurality of monocytes from the bone marrow;   (c) labeling the monocytes with biotin-conjugated lineage panel antibodies to one or more lineage surface antigens selected from the group consisting of CD2, CD3, CD4, CD11, CD11a, Mac-1, CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, Ly-6G, TER-119, CD45RA, CD56, CD64, CD68, CD86, CD66b, HLA-DR, and CD235a;   (d) removing monocytes that are positive for said one or more lineage surface antigens from the plurality of monocytes and recovering a population of lineage negative hematopoietic stem cells containing endothelial progenitor cells.   
     
     
         2 . The method of  claim 1  wherein the mammal is a human. 
     
     
         3 . The method of  claim 1  wherein the mammal is a human and the monocytes are labeled in step (c) with biotin-conjugated lineage panel antibodies to CD2, CD3, CD4, CD11a, Mac-1, CD14, CD16, CD19, CD33, CD38, CD45RA, CD64, CD68, CD86, and CD235a. 
     
     
         4 . The method of  claim 2  wherein the mammal is a human and the method includes the additional steps of labeling the monocytes with a biotin-conjugated CD133 antibody and recovering a population of CD133 positive, lineage negative hematopoietic stem cells. 
     
     
         5 . The method of  claim 2  wherein the mammal is a human and the method includes the additional steps of labeling the monocytes with a biotin-conjugated CD133 antibody, removing CD133 positive cells, and recovering a population of CD133 negative, lineage negative hematopoietic stem cells. 
     
     
         6 . A hematopoietic stem cell population isolated by the method of  claim 1 . 
     
     
         7 . A method of enhancing retinal neovascularization in a mammal comprising intravitreally injecting lineage negative hematopoietic stem cell population of  claim 6  into the eye of a mammal in need of retinal neovascularization wherein the stem cells are derived from bone marrow of the same species of mammal as the species into whose eye the cells are injected. 
     
     
         8 . The method of  claim 7  wherein the stem cells are derived from the bone marrow of the same individual mammal into whose eye the stem cells are injected. 
     
     
         9 . The method of  claim 7  wherein the mammal is a human. 
     
     
         10 . A method of treating an ocular disease in a mammal comprising isolating from the bone marrow of the mammal a lineage negative hematopoietic stem cell population that includes endothelial progenitor cells by:
 (a) extracting bone marrow from a mammal suffering from an ocular disease;   (b) separating a plurality of monocytes from the bone marrow;   (c) labeling the monocytes with biotin-conjugated lineage panel antibodies to one or more lineage surface antigens selected from the group consisting of CD2, CD3, CD4, CD11, CD11a, Mac-1, CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, Ly-6G, TER-119, CD45RA, CD56, CD64, CD68, CD86, CD66b, HLA-DR, and CD235a;   (d) separating monocytes that are positive for said one or more lineage surface antigens from the plurality of monocytes and recovering a population of lineage negative hematopoietic stem cells containing endothelial progenitor cells;   and subsequently intravitreally injecting the isolated stem cells into an eye of the mammal in a number sufficient to ameliorate the effects of the disease.   
     
     
         11 . The method of  claim 10  wherein the number of stem cells is effective for repairing retinal damage of the mammal's eye. 
     
     
         12 . The method of  claim 10  wherein the number of stem cells is effective for stabilizing retinal neovasculature of the mammal's eye. 
     
     
         13 . The method of  claim 10  wherein the number of stem cells is effective for maturing retinal neovasculature of the mammal's eye. 
     
     
         14 . A transfected lineage negative hematopoietic stem cell population comprising a stem cell population of  claim 6  transfected with a gene that operably encodes a therapeutically useful peptide. 
     
     
         15 . The transfected stem cell population of  claim 14  wherein the therapeutically useful peptide is an anti-angiogenic peptide. 
     
     
         16 . The transfected stem cell population of  claim 14  wherein the anti-angiogenic peptide is a protein fragment. 
     
     
         17 . The transfected stem cell population of  claim 14  wherein the therapeutically useful peptide is a neurotrophic agent. 
     
     
         18 . The transfected stem cell population of  claim 17  wherein the neurotrophic agent is selected form the group consisting of nerve growth factor, neurotrophin-3, neurotrophin-4, neurotrophin-5, ciliary neurotrophic factor, retinal pigmented epithelium-derived neurotrophic factor, insulin-like growth factor, glial cell line-derived neurotrophic factor, and brain-derived neurotrophic factor. 
     
     
         19 . A method of inhibiting retinal angiogenesis in the eye of a mammal comprising intravitreally injecting a transfected stem cell population according to  claim 15  into the eye of the mammal. 
     
     
         20 . A method of inhibiting retinal neuronal degeneration in the eye of a mammal comprising intravitreally injecting a transfected stem cell population according to  claim 17  into the eye of the mammal. 
     
     
         21 . A transfected lineage negative hematopoietic stem cell population prepared by:
 (a) extracting bone marrow from an adult mammal;   (b) separating a plurality of monocytes from the bone marrow;   (c) labeling the plurality of monocytes with biotin-conjugated lineage panel antibodies to CD2, CD3, CD4, CD11, CD11a, Mac-1, CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, Ly-6G, TER-119, CD45RA, CD56, CD64, CD68, CD86, CD66b, HLA-DR, and CD235a;   (d) separating monocytes that are positive for said one or more lineage surface antigens from the plurality of monocytes and recovering a population of lineage negative hematopoietic stem cells containing endothelial progenitor cells; and   (e) transfecting the lineage negative hematopoietic stem cells recovered in step (d) with a polynucleotide that operably encodes a therapeutically useful peptide.   
     
     
         22 . The transfected stem cell population of  claim 21  wherein the therapeutically useful peptide is an anti-angiogenic peptide. 
     
     
         23 . The transfected stem cell population of  claim 21  wherein the therapeutically useful peptide is a neurotrophic agent. 
     
     
         24 . A method of delivering transgenes to the retinal vasculature of a mammal comprising intravitreally injecting a transfected lineage negative hematopoietic stem cell population of  claim 14  into the eye of the mammal. 
     
     
         25 . A method of delivering transgenes to the retinal vasculature of a mammal comprising intravitreally injecting a transfected lineage negative hematopoietic stem cell population of  claim 21  into the eye of the mammal. 
     
     
         26 . A method of rescuing neuronal networks in the eye of a mammal comprising intravitreally injecting a transfected lineage negative hematopoietic stem cell population of  claim 6  into the eye of the mammal. 
     
     
         27 . A method of rescuing blood vessels in the eye of a mammal comprising intravitreally injecting a transfected lineage negative hematopoietic stem cell population of  claim 6  into the eye of the mammal. 
     
     
         28 . A method of stimulating upregulation of anti-apoptotic genes in the eye of a mammal comprising intravitreally injecting a lineage negative hematopoietic stem cell population of  claim 6  into the eye of the mammal. 
     
     
         29 . A method of repairing ischemic tissue in the eye of a mammal comprising intravitreally injecting a lineage negative hematopoietic stem cell population of  claim 6  into the eye of the mammal. 
     
     
         30 . A method of targeted delivery of stem cells to astrocytes in the eye of a mammal comprising intravitreally injecting a lineage negative hematopoietic stem cell population of  claim 6  into the eye of the mammal. 
     
     
         31 . A method of targeted delivery of transgenes to astrocytes in the eye of a mammal comprising intravitreally injecting a lineage negative hematopoietic stem cell population of  claim 14  into the eye of the mammal.