title: Application of mutant JAK2V617F for in vitro generation of red blood cells.
abstract: BACKGROUND
  In vitro generation of red blood cells (RBCs) from hematopoietic stem cells (HSCs) has been reported, but the collection of 1 × 10(5) to 1 × 10(6) CD34+ cells present in cord and peripheral blood is too small for expansion to 1 × 10(12) cells in 1 unit of RBCs. We transduced JAK2V617F gene, the most common mutation with polycythemia vera (PV), into cord blood-derived CD34+ cells. This PV model was expected to increase cell proliferation without the addition of erythropoietin (EPO) in early phase of differentiation.
  
  
  METHODS
  Empty vector (control), wild-type JAK2 (wJAK2), and mutant JAK2V617F (mJAK2) were transduced into CD34+ cells using a lentivirus system. The CD34+ cells were then differentiated to the RBCs in a culture system. The cells were analyzed for cell number, differential count, and morphologic changes. Cultured RBCs were tested for oxygen equilibrium.
  
  
  RESULTS
  wJAK2- and mJAK2-transduced cells showed higher proliferation capacity until Day 21 than control cells; interestingly, only mJAK2-transduced cells were highly increased on Day 7 during EPO-free culture. However, both wJAK2- and mJAK2-tranduced cells had more delayed differentiation than control, but they had a higher portion of completely matured RBCs and orthochromatic erythroblasts. Furthermore, mJAK2-tranduced cells showed more differentiation into RBCs than wJAK2-transduced cells and they had a normal hemoglobin dissociation curve.
  
  
  CONCLUSIONS
  This is the first trial to use a PV erythropoiesis model for RBC differentiation from stem cells. The transduction of HSCs with mJAK2 increased their proliferation capacity in EPO-free culture conditions. This model may also be useful for investigating the pathogenesis of PV.
authors.affiliation: Blood Transfusion Research Institute, Korean Red Cross, Wonju, Korea.
authors.forename: Sun Ah
authors.initials: SA
authors.lastname: Lee
authors.affiliation: Department of Laboratory Medicine, Yonsei University College of Medicine.
authors.forename: Ji Yeon
authors.initials: JY
authors.lastname: Kim
authors.affiliation: Department of Laboratory Medicine, Yonsei University College of Medicine.
authors.forename: Yongwook
authors.initials: Y
authors.lastname: Choi
authors.affiliation: Department of Laboratory Medicine, the Catholic University of Korea, Seoul St. Mary's Hospital, Seoul, Korea.
authors.forename: Yonggoo
authors.initials: Y
authors.lastname: Kim
authors.affiliation: Department of Laboratory Medicine, Yonsei University College of Medicine.
authors.forename: Hyun Ok
authors.initials: HO
authors.lastname: Kim
mesh_terms: D019943:Amino Acid Substitution; D018952:Antigens, CD34 / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics; D049109:Cell Proliferation / Q000235:genetics; D002478:Cells, Cultured; D004912:Erythrocytes / Q000378:metabolism*; D004920:Erythropoiesis / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics*; D050505:Mutant Proteins / Q000235:genetics; D010649:Phenylalanine / Q000235:genetics; D014162:Transfection; D014633:Valine / Q000235:genetics
chemical_list: D018952:Antigens, CD34; D050505:Mutant Proteins; D010649:Phenylalanine; C507924:JAK2 protein, human; D053614:Janus Kinase 2; D014633:Valine