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Application of mutant JAK2V617F for in vitro generation of red blood cells

 Sun Ah Lee  ;  Ji Yeon Kim  ;  Yongwook Choi  ;  Yonggoo Kim  ;  Hyun Ok Kim 
 Transfusion, Vol.56(4) : 837-843, 2016 
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Issue Date
Amino Acid Substitution* ; Antigens, CD34/metabolism ; Cell Differentiation/genetics ; Cell Proliferation/genetics ; Cells, Cultured ; Erythrocytes/metabolism* ; Erythropoiesis/genetics* ; Hematopoietic Stem Cells/metabolism ; Hematopoietic Stem Cells/physiology* ; Humans ; Janus Kinase 2/genetics* ; Mutant Proteins/genetics ; Phenylalanine/genetics ; Transfection* ; Valine/genetics
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. STUDY DESIGN AND 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. CONCLUSION: 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.
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1. College of Medicine (의과대학) > Dept. of Laboratory Medicine (진단검사의학교실) > 1. Journal Papers
Yonsei Authors
김현옥(Kim, Hyun Ok) ORCID logo https://orcid.org/0000-0002-4964-1963
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