Transcription Factor Mediated Programming Towards Megakaryocytes

Abstract

This invention relates to the forward programming of pluripotent stem cells (PSCs) into megakaryocyte (MK) progenitor cells using the transcription factors GATA1, FLI1 and TAL1. Methods of producing megakaryocyte (MK) progenitor cells and subsequently differentiating them into mature megakaryocytes are provided.

1 - 41 . (canceled) 
     
     
         42 . A method for producing platelets, the method comprising:
 (i) introducing into an isolated population of pluripotent stem cells (PSCs):
 (a) a first transgene comprising an inducible promoter and nucleotide sequences encoding GATA binding protein 1 (GATA1), Friend leukemia virus integration 1 (FLI1) and T cell acute lymphocytic leukemia protein 1 (TAL1) proteins for their concurrent transcription under the control of the inducible promoter; and 
 (b) a second transgene comprising a constitutive promoter and a nucleotide sequence encoding a transcription factor for the constitutive expression of the transcription factor under the control of the constitutive promoter, the transcription factor specific for binding to the inducible promoter in the first transgene thereby concurrently inducing the transcription of the GATA1, FLI1 and TAL1 proteins; and 
   (ii) culturing the pluripotent stem cells in ultralow adherent culture conditions in a chemically defined medium (CMD) comprising at least a chemical agent, BMP4, and FGF2 to produce megakaryocyte progenitor cells for a duration wherein the megakaryocyte progenitor cells produce platelets through pro-platelet formation.   
     
     
         43 . The method of  claim 42  wherein the BMP4 and FGF2 are recombinant human proteins. 
     
     
         44 . The method of  claim 42  wherein the inducible promoter is a tetracycline responsive promoter. 
     
     
         45 . The method of  claim 44  wherein the GATA1, FLI1 and TAL1 are concurrently transcribed under the influence of the tetracycline responsive promoter. 
     
     
         46 . The method of  claim 42  wherein the constitutively expressed transcription factor is a transactivator protein. 
     
     
         47 . The method of  claim 46  wherein the transactivator protein is a reverse tetracycline-controlled transactivator protein that binds to the tetracycline responsive promoter. 
     
     
         48 . The method of  claim 42  wherein the chemical agent is tetracycline or a derivative thereof. 
     
     
         49 . The method of  claim 42 , wherein the first and the second transgenes integrate at different sites in a chromosome of the pluripotent stem cells. 
     
     
         50 . The method of  claim 42 , wherein the pluripotent stem cells are selected from the group consisting of embryonic stem (ES) cells, non-embryonic stem cells, fetal or adult somatic stem cells and stem cells derived from non-pluripotent cells. 
     
     
         51 . The method of  claim 42  wherein said platelets are used as a therapeutic agent in platelet-associated conditions. 
     
     
         52 . The method of  claim 42  wherein the CDM further comprises thrombopoietin protein (TPO), and one or more of Stem Cell Factor (SCF) or IL1B. 
     
     
         53 . The method of  claim 52  wherein the TPO, SCF and IL1B are recombinant human proteins. 
     
     
         54 . The method of  claim 42  wherein the induced stem cells are cultured for a period of 18 to 22 days to produce the platelets from the megakaryocyte cells. 
     
     
         55 . The method of  claim 42  wherein the induced stem cells are cultured for a period of about 20 days to produce the platelets from the megakaryocyte cells. 
     
     
         56 . The method of  claim 54  wherein at least 95% of the platelet producing megakaryocyte cells express CD41a. 
     
     
         57 . The method of  claim 54  wherein at least 50% of the platelet producing megakaryocyte cells express CD42a. 
     
     
         58 . The method of  claim 54  wherein the platelet producing megakaryocyte cells optionally produce platelet like particles (PLPs). 
     
     
         59 . The method of  claim 54  wherein the pluripotent stem cells are induced pluripotent cells (iPS). 
     
     
         60 . The method of  claim 59  wherein the iPS cells are human iPS cells. 
     
     
         61 . The method of  claim 42  wherein the chemical agent is added transiently or constitutively to the culture conditions.