Cd34+cd41dim megakaryocytes progenitors and uses thereof for producing proplatelet-bearing mks and/or platelets

Abstract

The invention relates to a method of producing CD34 + CD41 dim megakaryocyte (MK) progenitor cells, and a substantially pure cell population of megakaryocyte precursor cells obtained by said method and compositions thereof. The invention also relates to a method of producing proplatelet-bearing MKs and/or platelets using the CD34 + CD41 dim cells.

What is claimed is: 
     
         1 . A substantially pure cell population of megakaryocyte (MK) progenitors wherein at least 80% of the cells in the population are CD34 + CD41 dim  cells. 
     
     
         2 . The cell population according to  claim 1 , wherein the cell population comprises at least 150,000 CD34 + CD41 dim  cells. 
     
     
         3 . The cell population according to  claim 1 , wherein the CD34 + CD41 dim  cells are CD34 + CD9 − CD41 + . 
     
     
         4 . The cell population according to  claim 1 , wherein said cell population is obtainable by a method of producing megakaryocyte (MK) progenitor cells comprising:
 a0) culturing haematopoietic stem cells (HSC) in a serum-free culture medium comprising low-density lipoprotein (LDL), stem cell factor (SCF), TPO, IL-6 and IL-9, in presence of an aryl hydrocarbon receptor (AhR) antagonist or by co-culture with human mesenchymal stromal cells (hMSCs), for a time sufficient to obtain a cell population comprising CD34 + CD41 dim  cells; and   a1) isolating said CD34 + CD41 dim  cells from said cell population.   
     
     
         5 . The cell population according to  claim 4 , wherein in a0) the cell population comprising CD34 + CD41 dim  cells is a cell population comprising CD34 + CD9 − CD41 dim  and wherein in a1) said CD34 + CD9 − CD41 +  cell population is isolated. 
     
     
         6 . A composition comprising a cell population of proplatelet-bearing megakaryocytes (MKs) and/or platelets and an infusion buffer for use for transfusion, wherein said use comprises preparing proplatelet-bearing megakaryocytes (MKs) and/or platelets by an ex vivo method of producing proplatelet-bearing megakaryocytes (MKs) and/or platelets comprising:
 a) culturing an isolated CD34 + CD41 dim  cell population of MK progenitors in a serum-free culture medium comprising thrombopoietin (TPO), in presence of an aryl hydrocarbon receptor (AhR) antagonist or by co-culture with human mesenchymal stromal cells (hMSCs), for a time sufficient to obtain a cell population comprising proplatelet-bearing MKs and/or platelets; and   b) collecting said cell population comprising proplatelet-bearing MKs and/or platelets,   which further comprises washing the proplatelet-bearing megakaryocytes (MKs) and/or platelets and suspending the washed cells in an infusion buffer.   
     
     
         7 . A method of manufacture of a medicament for transfusion to a patient in need thereof, comprising using a composition according to  claim 6 . 
     
     
         8 . A method of manufacture of a medicament for transfusion to a patient in need thereof, comprising using the cell population according to  claim 1 . 
     
     
         9 . A method of transfusion of a patient in need thereof, which comprises:
 a) preparing a composition comprising a cell population of proplatelet-bearing megakaryocytes (MKs) and/or platelets and an infusion buffer by an ex vivo method of producing proplatelet-bearing megakaryocytes (MKs) and/or platelets comprising:
 i) culturing an isolated CD34 + CD41 dim  cell population of MK progenitors in a serum-free culture medium comprising thrombopoietin (TPO), in presence of an aryl hydrocarbon receptor (AhR) antagonist or by co-culture with human mesenchymal stromal cells (hMSCs), for a time sufficient to obtain a cell population comprising proplatelet-bearing MKs and/or platelets; and 
 ii) collecting said cell population comprising proplatelet-bearing MKs and/or platelets, 
   which further comprises washing the proplatelet-bearing megakaryocytes (MKs) and/or platelets and suspending the washed cells in an infusion buffer   b) transfusing said composition to a patient in need thereof.   
     
     
         10 . A method according to  claim 9 , wherein the composition is administered by intravenous infusion and comprises at least 10 8  platelets/kg.