Korean J Hematol 2000; 35(2):
Published online June 30, 2000
© The Korean Society of Hematology
장기 액상배양을 이용한 제대혈 CD34＋ 세포의 체외증폭시 지지세포의 형성
유은선, 성주명
이화여자대학교 의과대학 소아과학교실,
이화여자대학교 의과대학 혈액종양내과학교실
Establishment of Adherent Cell Layers from Human Cord Blood CD34＋ Cells During Long-Term Liquid Culture Using TPO, FL, and G-CSF
BACKGROUND: The stromal cells, which one part of the microenvironment, including adipocyte, fibroblasts, endothelial cells, macrophage and smooth muscle cells provide a suitable environment for self-renewal, proliferation and differentiation of hematopoietic stem cells, as well as providing other regulators, cytokines to stimulate and enhance hematopoiesis. Unlike bone marrow, the in vitro development of this stroma cells from the umbilical cord blood (UCB) is not well established and has met very limited success. To see if stromal elements capable of supporting hematopoiesis could be grown from CD34＋ enriched umbilical cord blood, we was done stroma-free liquid culture system with thrombopoietin (TPO), flt3-ligand (FL) and the granulocyte-colony stimulating factor (G-CSF) using cord blood CB CD34＋ cells.
METHODS: Purified umbilical cord blood CD34＋ cells were cultured for five weeks in a stroma-free liquid culture system using TPO, FL, and G-CSF in the density of 1×10 5 cells/mL. When a firmly adherent monolayer of fibroblast like cells were fully formed, we evaluate morphology, immunophenotype, immunohistochemistry and functional assay, including non-adherent expansion, clonogenic progenitor expansion and apoptosis of established adherent cell layers.
RESULTS: By the 4th∼5th week, we found that firmly adherent fibroblast-like cells were established. These cells showed characteristics of endothelial cells expressing with VWF, VCAM-1, ICAM-1, CD31 and E-selectin, Furthermore, comparing without established endothelial monolayer, with established endothelial monolayer better expansion of total nucleated cells, CD34＋ cells and colony forming units granulocyte-macrophage (CFU-GM) during culture were found.
CONCLUSION: These results suggest that the UCB CD34＋ cell fraction contains stromal cell precursors and can establish the hematopoietic microenvironment to expand and sustain UCB CD34＋ cells through down-regulating apoptosis.
Keywords Cord blood CD34; EX vivc expansion; Adherent endothelial cell; Apoptosis; Long-term liquid culture;
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Korean J Hematol 2000; 35(2): 126-133
Published online June 30, 2000
Copyright © The Korean Society of Hematology.
장기 액상배양을 이용한 제대혈 CD34＋ 세포의 체외증폭시 지지세포의 형성
유은선, 성주명
이화여자대학교 의과대학 소아과학교실,
이화여자대학교 의과대학 혈액종양내과학교실
Establishment of Adherent Cell Layers from Human Cord Blood CD34＋ Cells During Long-Term Liquid Culture Using TPO, FL, and G-CSF
Eun Sun Yoo, Chu Myong Seong
Department of Pediatrics, Internal Medicine, College of Medicine, Ewha Womans University, Seoul, Korea
Abstract
BACKGROUND: The stromal cells, which one part of the microenvironment, including adipocyte, fibroblasts, endothelial cells, macrophage and smooth muscle cells provide a suitable environment for self-renewal, proliferation and differentiation of hematopoietic stem cells, as well as providing other regulators, cytokines to stimulate and enhance hematopoiesis. Unlike bone marrow, the in vitro development of this stroma cells from the umbilical cord blood (UCB) is not well established and has met very limited success. To see if stromal elements capable of supporting hematopoiesis could be grown from CD34＋ enriched umbilical cord blood, we was done stroma-free liquid culture system with thrombopoietin (TPO), flt3-ligand (FL) and the granulocyte-colony stimulating factor (G-CSF) using cord blood CB CD34＋ cells.
METHODS: Purified umbilical cord blood CD34＋ cells were cultured for five weeks in a stroma-free liquid culture system using TPO, FL, and G-CSF in the density of 1×10 5 cells/mL. When a firmly adherent monolayer of fibroblast like cells were fully formed, we evaluate morphology, immunophenotype, immunohistochemistry and functional assay, including non-adherent expansion, clonogenic progenitor expansion and apoptosis of established adherent cell layers.
RESULTS: By the 4th∼5th week, we found that firmly adherent fibroblast-like cells were established. These cells showed characteristics of endothelial cells expressing with VWF, VCAM-1, ICAM-1, CD31 and E-selectin, Furthermore, comparing without established endothelial monolayer, with established endothelial monolayer better expansion of total nucleated cells, CD34＋ cells and colony forming units granulocyte-macrophage (CFU-GM) during culture were found.
CONCLUSION: These results suggest that the UCB CD34＋ cell fraction contains stromal cell precursors and can establish the hematopoietic microenvironment to expand and sustain UCB CD34＋ cells through down-regulating apoptosis.
Keywords: Cord blood CD34, EX vivc expansion, Adherent endothelial cell, Apoptosis, Long-term liquid culture,
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