Stem cells derived from bone marrow for tissue regeneration

Abstract

The invention relates to stems cells derived from bone marrow, and uses thereof in tissue regeneration.

1 . A buffy coat obtainable by a method for separating bone marrow cells comprising the steps of:
 a) first centrifuging the bone marrow;   b) first collecting a first buffy coat layer and also collecting a layer of red blood cells from directly beneath the first buffy coat formed after step a) wherein the volume of red blood cells beneath the first buffy coat layer collected is between 1 to 20 times that of the first buffy coat layer;   c) second centrifuging the first buffy coat with the volume of red blood cells collected at step b); and   d) second collecting a second buffy coat layer formed after step c).   
     
     
         2 . The buffy coat according to  claim 1 , wherein steps a) and c) are carried out at 1500 G. 
     
     
         3 . The buffy coat according to  claim 1 , wherein steps a) and c) are carried out at 18° C. 
     
     
         4 . The buffy coat according to  claim 1 , wherein step a) is carried out for at least about 20 minutes and step c) is carried out for at least about 10 minutes, preferably for up to 20 minutes and more preferably for at least about 20 minutes. 
     
     
         5 . The buffy coat according to  claim 1 , wherein the method comprises additional steps between step b) and c) comprising the steps of:
 b1) collecting into at least one separate container a volume of remaining red blood cells left over after the removal of the first buffy coat layer and volume of red blood cells collected at step b);   c1) centrifuging the volume of red blood cells collected at step b1);   d1) collecting a third buffy coat layer formed after step c1); and   e1) mixing the third buffy coat collected after step d1) with the second buffy coat collected after step d).   
     
     
         6 . The buffy coat according to  claim 1 , wherein:
 the first centrifuging comprises separating components of the bone marrow to form a first layered product comprising the first buffy coat layer formed between a first red blood cell layer and a first plasma layer; and the first collecting comprises removing the first buffy coat layer from the first layered product.   
     
     
         7 . The buffy coat according to  claim 6 , wherein:
 the second centrifuging comprises forming a second layered product comprising the second buffy coat layer; and the second collecting comprises removing the second buffy coat layer from the second layered product.   
     
     
         8 . The buffy coat according to  claim 7 , wherein the removing the first buffy coat layer from the first layered product comprises: selectively removing at least a portion of the first plasma layer from adjacent the first buffy coat layer in the first layered product; and thereafter selectively removing the first buffy coat layer from adjacent to at least a portion of a red blood cell layer. 
     
     
         9 . The buffy coat according to  claim 6 , wherein steps a) and c) are carried out at 1500 G. 
     
     
         10 . The buffy coat according to  claim 6 , wherein steps a) and c) are carried out at 18° C. 
     
     
         11 . The buffy coat according to  claim 1 , wherein the second centrifuging is performed with the first buffy coat layer pooled in a container with other buffy coat layers collected during separately performing steps a) and b) on other bone marrow. 
     
     
         12 . The buffy coat according to  claim 1 , wherein cells contained in the buffy coat express CD 45 or CD 34. 
     
     
         13 . The buffy coat according to  claim 12 , wherein the cells contained in the buffy coat are stem cells. 
     
     
         14 . The buffy coat according to  claim 13 , wherein the cells contained in the buffy coat are of the haematopoietic or mesenchymal lineage. 
     
     
         15 . The buffy coat according to  claim 12 , wherein the concentration of cells contained in the buffy coat is 20-200 million per 2 to 10 ml, and wherein 2-20 million of these 20-200 million cells express CD 34. 
     
     
         16 . Method of promoting tissue regeneration in a patient by administering the buffy coat according to  claim 15 . 
     
     
         17 . Method of treatment of central nervous system disease or injury by administering the buffy coat according to  claim 15 . 
     
     
         18 . Method of treatment according to  claim 17 , wherein the disease or injury is selected from the group consisting of spinal cord injury, multiple sclerosis, cerebral vascular accident and amyotrophic lateral sclerosis. 
     
     
         19 . Method of treatment for healing a skin wound in a patient by administering the buffy coat according to  claim 15 . 
     
     
         20 . Method of treatment according to  claim 19 , wherein the skin wound is selected from the group consisting of skin wounds resulting from diabetes, ulcers, surgery, cosmetic surgery and burns. 
     
     
         21 . Method of promoting angiogenesis in a patient by administering the buffy coat according to  claim 15 . 
     
     
         22 . Method of treatment of erectile dysfunction in a patient by administering the buffy coat according to  claim 15 . 
     
     
         23 . Method of treatment of type 1 or type 2 diabetes in a patient by administering the buffy coat according to  claim 15 . 
     
     
         24 . Method of treatment of arthritis in a patient by administering the buffy coat according to  claim 15 . 
     
     
         25 . Method of treatment of liver damage in a patient by administering the buffy coat according to  claim 15 . 
     
     
         26 . Method of treatment of urinary incontinence in a patient by administering the buffy coat according to  claim 15 .