Isolation of human neural stem cells from amniotic fluid of patients with neural tube defects

Abstract

The present invention provides a method for isolating human neural stem cells from amniotic fluid of a patient whose fetus has been diagnosed to have a neural tube defect. Use of the isolated human neural stem cells in the treatment of neurological disorders is also provided.

What is claimed is: 
     
         1 . A method for treating a neurological condition in a mammal in need thereof, comprising administering an isolated human neural stem cells or a pharmaceutical composition comprising the isolated human neural stem cells to the mammal,
 wherein the isolated human neural stem cells are obtained from a method consisting of:   (a) collecting the cells from amniotic fluid obtained from a pregnant human subject whose fetus has been diagnosed to have a neural tube defect;   (b) incubating the cells with a culture medium;   (c) detecting expression levels of Nestin, Sox2, Musashi-1, and ATP-binding cassette G2 (ABCG2) markers and activity of Stage-Specific Embryonic Antigen-3 (SSEA-3), Stage-Specific Embryonic Antigen-4 (SSEA-4), Tumor Rejection Antigen-1-60 (TRA-1-60), TRA-1-81 and telomerase in the cells; and   (d) isolating from the culture medium human neural stem cells that express Nestin, Sox2, Musashi-1 and ABCG2 markers and exhibit telomerase activity but do not express SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 from the culture medium.   
     
     
         2 . The method of  claim 1 , wherein the neurological condition is a neurological disease with pathophysiological mechanisms involving ischemia, a neurological disease with pathophysiological mechanisms involving hypoxia, a neurodegenerative disease, or a disease of the nervous system accompanied by neural death. 
     
     
         3 . The method of  claim 1 , wherein the neural tube defect is anencephaly or myelomeningocele. 
     
     
         4 . The method of  claim 3 , wherein the neural tube defect is anencephaly. 
     
     
         5 . The method of  claim 1 , wherein the culture medium is a serum-free cell culture medium that allows the neural stem cells to proliferate. 
     
     
         6 . A method for screening drug candidates comprising: contacting an isolated human neural stem cells with a drug candidate; and determining one or more cell conditions of the cells, if the determined one or more cell conditions are better than the same condition(s) of the cells without contacting the drug candidate, it represents that the drug candidate has potential in the treatment of neurological conditions,
 wherein the isolated human neural stem cells are obtained from a method consisting of:   (a) collecting the cells from amniotic fluid obtained from a pregnant human subject whose fetus has been diagnosed to have a neural tube defect;   (b) incubating the cells with a culture medium;   (c) detecting expression levels of Nestin, Sox2, Musashi-1, and ATP-binding cassette G2 (ABCG2) markers and activity of Stage-Specific Embryonic Antigen-3 (SSEA-3), Stage-Specific Embryonic Antigen-4 (SSEA-4), Tumor Rejection Antigen-1-60 (TRA-1-60), TRA-1-81 and telomerase in the cells; and   (d) isolating from the culture medium human neural stem cells that express Nestin, Sox2, Musashi-1 and ABCG2 markers and exhibit telomerase activity but do not express SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 from the culture medium.   
     
     
         7 . The method of  claim 6 , wherein the neural tube defect is anencephaly or myelomeningocele. 
     
     
         8 . The method of  claim 7 , wherein the neural tube defect is anencephaly. 
     
     
         9 . The method of  claim 6 , wherein the culture medium is a serum-free cell culture medium that allows the neural stem cells to proliferate. 
     
     
         10 . A method for testing cytotoxicity of a drug candidate comprising: contacting an isolated human neural stem cells with the drug candidate; and determining one or more cell conditions of the cells, if the determined one or more cell conditions are poorer than the same condition(s) of the cells without contacting the drug candidate, it represents that the drug candidate may have cytotoxicity,
 wherein the isolated human neural stem cells are obtained from a method consisting of:   (a) collecting the cells from amniotic fluid obtained from a pregnant human subject whose fetus has been diagnosed to have a neural tube defect;   (b) incubating the cells with a culture medium;   (c) detecting expression levels of Nestin, Sox2, Musashi-1, and ATP-binding cassette G2 (ABCG2) markers and activity of Stage-Specific Embryonic Antigen-3 (SSEA-3), Stage-Specific Embryonic Antigen-4 (SSEA-4), Tumor Rejection Antigen-1-60 (TRA-1-60), TRA-1-81 and telomerase in the cells; and   (d) isolating from the culture medium human neural stem cells that express Nestin, Sox2, Musashi-1 and ABCG2 markers and exhibit telomerase activity but do not express SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 from the culture medium.   
     
     
         11 . The method of  claim 10 , wherein the neural tube defect is anencephaly or myelomeningocele. 
     
     
         12 . The method of  claim 11 , wherein the neural tube defect is anencephaly. 
     
     
         13 . The method of  claim 10 , wherein the culture medium is a serum-free cell culture medium that allows the neural stem cells to proliferate.