Methods for collecting and using placenta cord blood stem cells

Abstract

An innovative method of collecting cord blood stem cells from an isolated mammalian non-exsanguinated or partially exsanguinated placenta by placental perfusion is described and also an easy method for safe long duration cold storage of the placenta. Placental perfusion can include perfusing the isolated placenta with a pulsatile flow of perfusion solution, for example, using a pulsatile or peristaltic pump or device. The stem cells can then be isolated from the perfusate. Significantly increased amounts of CD133+ stem cells can be collected from the perfusate. The perfusion solution can include an anticoagulant. The isolated mammalian placenta need not be treated with an anticoagulant prior to perfusing. The isolated placenta can be free from an anticoagulant prior to perfusing.

What is claimed is: 
     
         1 . A method for treating a mammal in need of hematopoietic reconstitution or regeneration of damaged tissue, comprising:
 a) providing isolated stem cells by:
 i) performing machine perfusion of an isolated mammalian placenta comprising cord blood comprising cord blood stem cells,
 said performing machine perfusion being done with a pressure mediated pulsatile flow of a perfusion solution to produce a perfusate comprising cord blood comprising cord blood stem cells, 
 wherein said isolated mammalian placenta is non-exsanguinated; 
 
 ii) collecting the perfusate; and 
 iii) isolating the cord blood stem cells from the perfusate to produce isolated cord blood stem cells; and 
   b) introducing into the mammal a composition comprising a therapeutically effective amount of cells selected from the group consisting of the isolated stem cells and cells derived therefrom.   
     
     
         2 . A method according to  claim 1 , wherein said introducing comprises:
 introducing into the mammal a composition comprising a therapeutically effective amount of the isolated stem cells.   
     
     
         3 . A method according to  claim 1 , wherein said introducing comprises:
 obtaining said cells derived therefrom by culturing in vitro the isolated stem cells, thereby producing said cells derived therefrom; and   introducing into the mammal a composition comprising a therapeutically effective amount of said cells derived therefrom.   
     
     
         4 . The method according to  claim 3 , wherein said cells derived therefrom are differentiated cells or expanded stem cells. 
     
     
         5 . The method according to  claim 1 , wherein:
 the mammal is in need of hematopoietic reconstitution; and   said isolated stem cells comprise hematopoietic stem cells.   
     
     
         6 . The method according to  claim 5 , wherein the mammal in need of hematopoietic reconstitution has aplastic anemia, a hematopoietic malignancy, an autoimmune disease, a genetic disorder, an immunodeficiency, a malignant solid tumor, or a combination thereof. 
     
     
         7 . The method according to  claim 6 , in which the mammal in need of hematopoietic reconstitution has a hematopoietic malignancy selected from leukemia, lymphoma, multiple myeloma, and myelodysplastic syndrome. 
     
     
         8 . The method according to  claim 6 , in which the mammal in need of hematopoietic reconstitution has an immunodeficiency resulting from irradiation, chemotherapy, infection by a pathogenic microorganism, or a combination thereof. 
     
     
         9 . The method according to  claim 5 , wherein said introducing comprises introducing into the mammal intravenously or intraosseously a composition comprising a therapeutically effective amount of cells selected from the group consisting of the isolated stem cells and cells derived therefrom. 
     
     
         10 . The method according to  claim 1 , wherein:
 the mammal is in need of regeneration of damaged tissue.   
     
     
         11 . The method according to  claim 10 , wherein:
 said introducing comprises introducing into the mammal intravenously or by direct injection into the target organ a composition comprising a therapeutically effective amount of cells selected from the group consisting of isolated stem cells and cells derived therefrom   
     
     
         12 . The method according to  claim 11 , wherein the tissue comprises one or more of cardiac tissue, muscle tissue, liver, skin, neural tissue, bone tissue, epithelia, stroma, or endothelium. 
     
     
         13 . A method for treating a mammal in need of hematopoietic reconstitution or regeneration of damaged tissue, comprising:
 (a) optionally culturing in vitro isolated cord blood stem cells, thereby producing differentiated cells or expanded stem cells,
 said isolated cord blood stem cells being obtained by: 
 i) performing machine perfusion of an isolated mammalian placenta comprising cord blood comprising cord blood stem cells,
 said performing machine perfusion being done with a pressure mediated pulsatile flow of a perfusion solution to produce a perfusate comprising cord blood comprising cord blood stem cells, 
 wherein said isolated mammalian placenta is non-exsanguinated; 
 
 ii) collecting the perfusate; and 
 iii) isolating the cord blood stem cells from the perfusate to produce isolated cord blood stem cells; and 
   (b) introducing into the mammal a composition comprising a therapeutically effective amount of cells selected from the group consisting of:
 i) said isolated cord blood stem cells; 
 ii) said differentiated cells or expanded stem cells, and 
 iii) combinations thereof; 
   whereby hematopoietic reconstitution or tissue regeneration is effected.   
     
     
         14 . A method for treating a mammal in need of hematopoietic reconstitution or regeneration of damaged tissue, comprising:
 a) providing isolated stem cells by:
 i) providing an isolated mammalian placenta comprising cord blood comprising cord blood stem cells, wherein said isolated mammalian placenta is partially exsanguinated to produce a volume of cord blood comprising a first population of isolated cord blood stem cells; 
 ii) conducting machine perfusion of the isolated mammalian placenta with a pressure mediated flow of a perfusion solution to produce a perfusate comprising cord blood comprising cord blood stem cells, wherein said machine perfusion is performed using a pump; 
 iii) collecting the perfusate; and 
 iv) isolating the cord blood stem cells from the perfusate to produce a second population of isolated cord blood stem cells;
 wherein said second population of isolated cord blood cells has a greater percentage of CD133+ cells than said first population of isolated cord blood cells; and 
 
   b) introducing into the mammal a composition comprising a therapeutically effective amount of cells from the second population of isolated cord blood stem cells or cells derived from the second population of isolated cord blood stem cells.   
     
     
         15 . A method according to  claim 14 , wherein said introducing comprises:
 introducing into the mammal a composition comprising a therapeutically effective amount of cells from the second population of isolated cord blood stem cells.   
     
     
         16 . A method according to  claim 14 , wherein said introducing comprises:
 obtaining said cells derived therefrom by culturing in vitro cells from the second population of isolated cord blood stem cells, thereby producing said cells derived from the second population of isolated cord blood stem cells; and   introducing into the mammal a composition comprising a therapeutically effective amount of said cells derived from the second population of isolated cord blood stem cells.   
     
     
         17 . A method according to  claim 14 , wherein said introducing comprises introducing into the mammal a composition comprising a therapeutically effective amount of cells from the second population of isolated cord blood stem cells,
 said composition further comprising cells from the first population of isolated cord blood stem cells.