Large scale generation of functional megakaryocytes and platelets from human embryonic stem cells under stromal-free conditions

Abstract

The present invention provides a method of generating megakaryocytes and platelets. In various embodiments, method involves the use of human embryonic stem cell derived hemangioblasts for differentiation into megakaryocytes and platelets under serum and stromal-free condition. In this system, hESCs are directed towards megakaryocytes through embryoid body formation and hemangioblast differentiation. Further provided is a method of treating a subject in need of platelet transfusion.

1 . A method of generating megakaryocytes, comprising:
 providing hemangioblasts; and   culturing the hemangioblasts to differentiate into megakaryocytes (MKs).   
     
     
         2 - 19 . (canceled) 
     
     
         20 . A quantity of megakaryocytes generated from the method of  claim 1 . 
     
     
         21 . A method of generating platelets, comprising:
 culturing human pluripotent stem cells to produce hemangioblasts:   differentiating the hemangioblasts to obtain megakaryocytes (MKs); and   culturing the MKs to differentiate into platelets.   
     
     
         22 - 27 . (canceled) 
     
     
         28 . The method of  claim 21 , wherein the pluripotent stem cells are human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs). 
     
     
         29 . (canceled) 
     
     
         30 . The method of  claim 21 , wherein the pluripotent stem cells are cultured to form embryoid bodies (EBs) and the EBs are further cultured to produce hemangioblasts. 
     
     
         31 - 32 . (canceled) 
     
     
         33 . The method of  claim 30 , wherein the formed EBs are chemically and/or mechanically dissociated and cultured in the presence of at least one growth factor selected from the group consisting of BMP-4, VEGF, bFGF, TPO, Flt3 ligand, and SCF to generate the hemangioblasts. 
     
     
         34 - 40 . (canceled) 
     
     
         41 . The method of  claim 21 , wherein the MKs are cultured for at least 4 days in medium comprising at least one growth factor selected from the group consisting of TPO, SCF, sodium heparin, and IL1 to differentiate into the platelets. 
     
     
         42 . The method of  claim 41 , wherein the concentration of TPO is 100 ng/ml, SCF is 50 ng/ml, sodium heparin is 25 units/ml, and IL11 is 20 ng/ml. 
     
     
         43 . The method of  claim 41 , further comprising culturing the MKs in the presence of GM 6001 or IL3 after the at least 4 days. 
     
     
         44 . The method of  claim 41 , further comprising replacing at least a portion of the medium with medium comprising the at least one growth factor at least every 2 days. 
     
     
         45 . The method of  claim 41 , where in the medium is IMDM medium. 
     
     
         46 . The method of  claim 21 , wherein the MKs are dissociated and cultured on a mitotically arrested feeder layer for at least 4 days in medium comprising at least one growth factor selected from the group consisting of TPO, SCF, sodium heparin, and IL11 to differentiate into the platelets. 
     
     
         47 . The method of  claim 46 , wherein the feeder layer comprises OP9 cells. 
     
     
         48 . The method of  claim 46 , wherein the feeder layer comprises C3H 10T1/2 cells. 
     
     
         49 . The method of  claim 46 , further comprising culturing the MKs in the presence of GM 6001 or IL3 after the at least 4 days. 
     
     
         50 . The method of  claim 46 , further comprising replacing at least a portion of the medium with medium comprising the at least one growth factor at least every 2 days. 
     
     
         51 . The method of  claim 46 , wherein the medium is IMDM medium. 
     
     
         52 . A quantity of platelets generated from the method of  claim 21 . 
     
     
         53 - 54 . (canceled) 
     
     
         55 . A method of screening for a modulator of cellular differentiation comprising:
 providing a quantity of megakaryocytes (MKs);   contacting the MKs with a test compound; and   determining the presence or absence of a functional effect from the contact between the MKs and the test compound,   wherein the presence of a functional effect indicates that the test compound is a megakaryopoietic, thrombopoietic, and/or hematopoeitic factor that modulates cellular differentiation and the absence of a functional effect indicates that the test compound is not a megakaryopoietic, thrombopoietic, and/or hematopoeitic factor that modulates cellular differentiation.   
     
     
         56 . A method of treating a subject in need of platelet transfusion, comprising:
 administering a quantity of the platelets generated by the method of  claim 21  to the subject in need of the platelet transfusion, thereby treating the subject in need of platelet transfusion.   
     
     
         57 . (canceled)