Internally administered therapeutic agents for cranial nerve diseases comprising mesenchymal cells as an active ingredient

Abstract

Intravenous administration of bone marrow cells collected from rat bone marrow or peripheral blood to a rat cerebral infarction model was found to be effective in treating cerebral infarction. Human and murine bone marrow stem cells showed similar effects. Mesenchymal cells such as bone marrow cells, cord blood cells, or peripheral blood cells can be used as agents for in vivo administration against cranial nerve diseases.

1 . A composition of isolated and purified peripheral blood-derived mesenchymal stem cells (PMSCs), wherein the PMSCs are over 70% positive for CD73, essentially 100% positive for CD90, essentially 100% negative for CD 45  and essentially 100% negative for CD106.  
     
     
         2 . A method preparing peripheral blood-derived mesenchymal stem cells (PMSCs) comprising: 
 a) obtaining peripheral blood from a subject or patient,    b) optionally diluting the peripheral blood,    c) optionally incubating the peripheral blood,    d) centrifuging the peripheral blood in order to form a supernatant and a cell fraction,    e) discarding the supernatant from the cell fraction,    f) suspending the cell fraction in a culture medium,    g) plating the suspended cells on a surface for tissue culturing,    h) incubating the suspended cells for about 48 hours, wherein the cells form into a group of cells adhering the surface and a group of cells not adhering to the surface,    i) eliminating the nonadherent cells,    j) further incubating the adherent cells,    k) optionally detaching and subculturing the adherent cells,    l) detaching the adherent cells,    m) suspending the adherent cells in a neural progenitor basal medium,    n) plating the cells wherein PMSCs are formed, and    o) harvesting the PMSCs.    
     
     
         3 . The method of  claim 2 , wherein the peripheral blood is diluted in a red blood cell lysis solution.  
     
     
         4 . The method of  claim 2 , wherein the peripheral blood is centrifuged at 300-500 G for about 2 to 5 minutes.  
     
     
         5 . The method of  claim 2 , wherein the angle of the centrifuge tube is 40- 50° and the speed of the centrifuge is between about 1200 to about 3500 rpm.  
     
     
         6 . The method of  claim 2 , wherein the PMSCs are over 70% positive for CD73, essentially 100% positive for CD 90 , essentially 100% negative for CD 45  and essentially 100% negative for CD 106 .  
     
     
         7 . A method of preparing peripheral blood-derived mesenchymal stem cells (PMSCs) comprising: 
 a) obtaining peripheral blood from a subject or patient,    b) optionally diluting the peripheral blood,    c) optionally incubating the peripheral blood,    d) centrifuging the peripheral blood in order to form a supernatant and a cell fraction,    e) discarding the supernatant from the cell fraction,    f) suspending the cell fraction in a culture medium,    g) plating the suspended cells on a surface for tissue culturing,    h) incubating the suspended cells for about 48 hours, wherein the cells form into a group of cells adhering the surface and a group of cells not adhering to the surface,    i) eliminating the nonadherent cells,    j) further incubating the adherent cells,    k) optionally detaching and subculturing the adherent cells,    l) detaching the adherent cells,    m) suspending the adherent cells in a neural progenitor basal medium,    n) plating the cells wherein PMSCs are formed,    o) incubating the cells while optionally adding growth factors, wherein floating neurospheres of PMSCs are formed,    p) collecting the neurospheres of PMSCs,    q) dissociating the of PMSCs cells of the neurospheres,    r) plating the dissociated neurospheres, and    s) culturing the PMSCs.    
     
     
         8 . The method of  claim 7 , wherein after the non-adherent cells are eliminated, the adherent cells are incubated until reaching or nearly reaching confluence.  
     
     
         9 . The method of  claim 7 , wherein the neurospheres are centrifuged at 300-500 G for about 2 to 5 minutes.  
     
     
         10 . The method of  claim 9 , wherein the neurospheres are centrifuged in a neural progenitor base medium.  
     
     
         11 . The method of  claim 7 , wherein the PMSCs are over 70% positive for CD73, essentially 100% positive for CD 90 , essentially 100% negative for CD 45  and essentially 100% negative for CD 106 .  
     
     
         12 . A method of treating a patient who has suffered a stroke or who suffers from neural lesions caused by injury or disease, ischemia, infarction, Krabbe's disease, Hurler's syndrome, metachromatic leukodystrophy, or encephalomyelitis by injecting into the patient a therapeutically effective amount of the cell fraction of  claim 1 .  
     
     
         13 . The method of  claim 12 , wherein the injection is intravenous.  
     
     
         14 . The method of  claim 13 , wherein the site of the injection is the patient's arm or leg.  
     
     
         15 . The method of  claim 12 , wherein the patient has suffered a stroke.  
     
     
         16 . The method of  claim 12 , wherein the encephalomyelitis is autoimmune encephalomyelitis.  
     
     
         17 . A method of treating a patient who has suffered a stroke or who suffers from neural lesions caused by injury or disease, ischemia, infarction, Krabbe's disease, Hurler's syndrome, metachromatic leukodystrophy, or encephalomyelitis by injecting into the patient a therapeutically effective amount of the cell fraction of  claim 2 .  
     
     
         18 . The method of  claim 17 , wherein the injection is intravenous.  
     
     
         19 . The method of  claim 18 , wherein the site of the injection is the patient's arm or leg.  
     
     
         20 . The method of  claim 17 , wherein the patient has suffered a stroke.  
     
     
         21 . The method of  claim 17 , wherein the encephalomyelitis is autoimmune encephalomyelitis. 1568834.2