Establishment of Adherent Cell Layers from Human Cord Blood CD34+ Cells During Long-Term Liquid Culture Using TPO, FL, and G-CSF / 대한혈액학회지

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 1x105 cells/mL. When a firmly adherent monolayer of fibroblast like cells were fully formed, we evaluate morphology, immunopheno-type, 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.

Immunophenotypic Analysis of Long-term Culture-Initiating Cells in Long-term Liquid Culture / 대한소아혈액종양학회지

Purpose: Long-term culture- initiating cells(LTC-IC) are stem cells that have the capacities of long-term engraftment and helping to establish hematopoietic microenvironment. For evaluation of the LTC-IC, we measured the counts and function with multidimentional flowcytometry in long-term culture media. METHODS: Samples were obtained from umbilical cord blood, leukapheresis products and bone marrow(BM). LTC-IC were counted with flowcytometric analysis using anti- CD34, anti-CD38, and anti-HLA-DR antibodies at 0, 5, and 8 weeks. Cell adhesion molecule related with stem cell were evaluated with flowcytometric analysis also using anti-VCAM-1(CD106) and anti-VLA-4(CD49d) at 0 and 8 weeks. RESULTS: The proportion of CD34+/CD38- cell from fractionated mononuclear cells at 0 week were 0.46%, 0.044%, and 0.038% for BM, leukapheresis products, and umbilical cord blood respectively and then rapidly decreased at 5 weeks, but still persisted at 8 weeks in all three groups. The proportion of CD34+/HLA-DR- cells was the same tendency to CD34+/CD38-. VCAM+ expression rate from fractionated CD34+ cells at 0 and 8 weeks were 67.3% and 40.2% for BM and 64.1% 44.2% for umbilical cord blood but it was very low 31.2% and 5.1% for leukapheresis products. VLA-4+ expression rate for fractionated CD34+ cells at 0 and 8 weeks were similar tendency to VCAM+ cells. CONCLUSION: This study suggest that the count of LTC-IC decreased with time but still persisted until 8 weeks. Umbilical cord blood including BM help to establish the hematopoietic microenvironments.