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KMID : 0391319990090020161
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Korean Journal of Biological Response Modifiers
1999 Volume.9 No. 2 p.161 ~ p.174
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Effect of Thrombopoietin on ex-vivo Megakaryopoiesis
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Cho Sang-Deog
Lee Kuk-Kyung
Bae Sang-Byung
Suh Won-Suk
Kim Sook-Ja
Jung Hee-Jung
Lee Kyu-Taek
Park Sung-Kyu
Won Jong-Ho
Baick Seung-Ho
Hong Dae-Sik
Park Hee-Sook
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Abstract
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Background : After decades of study, the regulation of megakaryocytopoiesis is still not well understood. However the recent cloning and characterization of thrombopoietin, a ligand for the receptor encoded by the c-mpl proto-oncogene, provides new insights into the humoral regulation of megakaryocytopoiesis and platelet production. The aims of the present study were to investigate the effects of human recombinant thrombopoietin alone or in combination with other several hematopoietic factors on megakaryocyte colony formation from human umbilical cord blood CD34+ cells and to investigate and elucidate the apoptotic cells induced by the n vitro withdrawal of thrombopoietin.
Methods : Human cord blood CD34+ cells were cultured in MegacultTM (Stem Cell Technologies Inc. Vancouver, Canada) in the presence of recombinant growth factors and CFU-Meg colonies were counted on day 14. CD34+ and CD41a+ cell expansion was analysed using serum free liquid culture system for 7 days with recombinant growth factors. Apoptosis was identified by in situ nick end labeling method.
Results : rhTPO alone had a concentration-dependent effect on megakaryocyte colony growth. At concentrations above 1ng/mL, rhTPO supported significant megakaryocyte colony formation in a concentration dependent manner. The combination of rhTPO plus other cytokines, including rhEPO, IL-3 and SCF (not IL-6) resulted in a synergistic
enhancement of both the number and size of megakaryocyte colonies. The number of CD41a+ cells were increased to 37.33±9.50% (1ng/mL), 57.12±8.63% (10ng/mL), and 59.17±4.41% (20ng/mL) of total expanded cells, respectively, during the culture of the purified CD34+ cells in liquid phase in the presence of various concentration of rhTPO
for 7 days. The number of CD41a+ cells wee increased to 33.01±13.76% (TPO+EPO), 31.41±10.80% (TPO+IL-3), 57.27±10.01 (TPO+IL-6), and 35.32±8.34% (TPO+SCF) of total expanded cells, respectively, during the culture of the purified CD34+ cells in liquid phase for 7 days in the presence of rhTPO (10ng/mL) with or without rhEPO, IL-3,
IL-6, or SCF. When the purified CD34+ cells were cultured with or without rhTPO, the number of viable CD34+ cells were decreased and that of apoptotic cells were increased after the deprivation of rhTPO.
Conclusion : The results of the present study indicate that TPO exerts a powerful proliferative activity toward CFU-MK from human umbilical cord blood, suggesting that TPO may be a primary regulator of megakaryocytopoiesis.
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KEYWORD
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Thrombopoietin, Megakaryocytopoiesis, Apoptosis,
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