Human Extensively Self-Renewing Erythroblasts (ESRE)

Abstract

The present invention provides a human cell population that can self-renew extensively and yet retain the capacity to differentiate into red blood cells (RBCs). These cells are referred to as extensively self-renewing erythroblasts (ESREs). The cells of the invention serve among other things as a renewable source of transfusable RBCs.

1 - 33 . (canceled) 
     
     
         34 . A genetically engineered human red blood cell (RBC) generated from an extensively self-renewing erythroblast (ESRE), wherein the ESRE has been made by a process comprising introducing an exogenous nucleic acid encoding Bmi-1, or a variant or fragment thereof, into a human stem cell. 
     
     
         35 . The genetically engineered human RBC of  claim 34 , comprising an exogenous therapeutic protein. 
     
     
         36 . The genetically engineered human RBC of  claim 35 , wherein the exogenous therapeutic protein is selected from the group consisting of factor VIII, factor VIIIa, factor V, factor Va, factor IX, factor X, factor XI, factor XII, factor XIII, von Willebrand's factor, tissue plasminogen activator, protein C, protein S, and antithrombin III. 
     
     
         37 . The genetically engineered human RBC of  claim 34 , comprising an exogenous detectable polypeptide. 
     
     
         38 . The genetically engineered human RBC of  claim 37 , wherein the detectable polypeptide is selected from the group consisting of a luciferase, a fluorescent protein, a phosphatase, a peroxidase, a kinase, chloramphenicol transferase, and beta-galactosidase. 
     
     
         39 . The genetically engineered human RBC of  claim 34 , wherein the RBC is generated by a process comprising culturing the ESRE in a differentiation medium comprising erythropoietin (EPO) and insulin. 
     
     
         40 . The genetically engineered human RBC of  claim 39 , wherein the process comprises culturing the cells in an expansion medium comprising EPO, stem cell factor (SCF), dexamethasone, and a lipid mixture. 
     
     
         41 . The genetically engineered human RBC of  claim 34 , wherein the stem cell is an adult stem cell. 
     
     
         42 . The genetically engineered human RBC of  claim 34 , wherein the stem cell is an embryonic stem cell. 
     
     
         43 . The genetically engineered human RBC of  claim 34 , wherein the stem cell is selected from the group consisting of a hematopoietic stem cell, an induced pluripotent stem (iPS) cell, a cord blood stem cell, a bone marrow stem cell, a placental stem cell, a circulating peripheral blood stem cell, and a reprogrammed stem cell. 
     
     
         44 . The genetically engineered human RBC of  claim 34 , wherein the introducing the exogenous nucleic acid comprises tranducing or transfecting the human stem cell. 
     
     
         45 . The genetically engineered human RBC of  claim 34 , wherein the RBC is a reticulocyte. 
     
     
         46 . The genetically engineered human RBC of  claim 34 , wherein the RBC is an erythrocyte. 
     
     
         47 . A liquid medium comprising a plurality of the human RBCs of  claim 34 , wherein the liquid medium is suitable for administration to a human subject. 
     
     
         48 . A method of treating a disease or disorder in a subject, the method comprising administering the human RBC of  claim 34  to the subject. 
     
     
         49 . A method of generating a genetically engineered human red blood cell (RBC), the method comprising culturing an extensively self-renewing erythroblast (ESRE) under conditions suitable for differentiation of the ESRE into a RBC, wherein the ESRE comprises an exogenous nucleic acid encoding Bmi-1 or a variant or fragment thereof made by a process comprising introducing said exogenous nucleic acid into a human stem cell. 
     
     
         50 . The method of  claim 49 , further comprising introducing an exogenous nucleic acid encoding a therapeutic protein or a detectable polypeptide into the human stem cell or progeny thereof. 
     
     
         51 . The method of  claim 49 , further comprising introducing an exogenous nucleic encoding a therapeutic protein or a detectable polypeptide into the ESRE or progeny thereof 
     
     
         52 . A method of generating an extensively self-renewing erythroblast (ESRE), comprising introducing an exogenous nucleic acid encoding Bmi-1, or a variant or fragment thereof, into a human stem cell. 
     
     
         53 . The method of  claim 52 , wherein the introducing the exogenous nucleic acid comprises viral delivery using an adenovirus, an adeno-associated virus, a herpesviruse, a lentivirus, a papillomavirus, or a retrovirus.