Isolation, expansion and use of clonogenic endothelial progenitor cells

Abstract

A hierarchy of endothelial colony forming cells (EPCs) was identified from mammalian cord blood, umbilical vein and aorta. A newly isolated cell named high proliferative potential—endothelial colony forming cell (HPP-ECFC) was isolated and characterized. Single cell assays were developed that test the proliferative and clonogenic potential of endothelial cells derived from cord blood, or from HUVECs and HAECs. EPCs were found to reside in vessel walls. Use of a feeder layer of cells derived from high proliferative potential-endothelial colony forming cells (HPP-ECPCS) from human umbilical cord blood, stimulates growth and survival of repopulating hematopoietic stem and progenitor cells. Stimulation of growth and survival was determined by increased numbers of progenitor cells in in vitro cultures and increased levels of human cell engraftment in the NOD/SCID immunodeficient mouse transplant system.

1 . Isolated endothelial colony forming cells that have the following characteristics: 
 (a) express cell surface antigens that are characteristic of endothelial cells;    (b) do not express cells surface antigens that are characteristic of hematopoietic cells;    (c) ingest acetylated LDL; and    (d) form capillary-like tubes in matrigel.    
   
   
       2 . The isolated cells of  claim 1  wherein the cell surface antigens characteristic of endothelial cells are CD31, CD105, CD144, and CD146.  
   
   
       3 . The isolated cells of  claim 1  wherein the cell surface antigens characteristic of hematopoietic cells are CD14 and CD45.  
   
   
       4 . The isolated cells of  claim 1  further defined as: 
 (a) replate into at least secondary colonies of at least about 2000 cells when plated from a single cell;    (b) exhibit high proliferation;    (c) proliferate from a single cell; and    (d) express high levels of telomerase.    
   
   
       5 . The cells of  claim 4  wherein high levels of telomerase expression are at least about 34% of that expressed by HeLa cells.  
   
   
       6 . The isolated cells of  claim 4  further defined as displaying a high nuclear to cytoplasmic ratio that is >0.8.  
   
   
       7 . The method of  claim 4  wherein at least 10 7  progeny derive from a single cell.  
   
   
       8 . The isolated cells of  claim 4  further defined as having a cell diameter <22 microns.  
   
   
       9 . A method of isolating the cells of  claim 1 , the method comprising: 
 (a) culturing cells from a biological sample on supports coated with extracellular matrix proteins;    (b) selecting cells that adhere to the supports and form replatable colonies; and    (c) selecting single cells from the colonies.    
   
   
       10 . The method of  claim 9  wherein the biological sample is mammalian blood.  
   
   
       11 . The method of  claim 9  wherein the biological sample is a mammalian blood vessel.  
   
   
       12 . A cell isolated by the method of  claim 9 .  
   
   
       13 . A single cell assay for types of endothelial cells comprising: 
 (a) cell sorting of biological samples using a specific sorting method;    (b) culturing the single sorted cells on extracellular matrix protein under defined conditions; and    (c) enumerating specific colony sizes, morphology, and proliferative potential to determine the type of endothelial cell.    
   
   
       14 . The single cell assay of  claim 13  wherein the types of endothelial cells comprise HPP-ECFC.  
   
   
       15 . A method of enriching for cells of  claim 12 , the method comprising: 
 (a) cell sorting of biological samples using a specific sorting method; and    (b) culturing of single sorted cells on extracellular matrix proteins under defined conditions.    
   
   
       16 . A method for expanding hematopoietic stem cells ex vivo, the method comprising: 
 (a) culturing HPP-ECFC cells on collagen coated solid supports; and    (b) expanding hematopoietic stem cells (HSC) by co-culturing with HPP-ECFC cells.    
   
   
       17 . The method of  claim 16 , wherein the HPP-ECFCs are derived from human cord blood cells and the HSC cells are derived from human bone marrow.  
   
   
       18 . A method for improving the percentage of hematopoietic stem cells in a graft in a mammal, the method comprising: 
 (a) co-culturing human bone marrow cells with cord blood HPP-ECFC to form a product; and    (b) transplanting a suitable amount of the product into the mammal.    
   
   
       19 . The method of  claim 18 , wherein the cells are CD45+cells derived from human bone marrow, and the mammal is a NOD-SCID mouse.