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,