Rossi Barbara, Zanolin Elisabetta, Vincenzi Carlo, Diani Franco, Pizzolo Giovanni, de Wynter Erika, Nadali Gianpaolo
Department of Clinical and Experimental Medicine, Section of Hematology, University of Verona, Italy.
Stem Cells Dev. 2004 Aug;13(4):362-71. doi: 10.1089/scd.2004.13.362.
The aim of this study was to clarify the mechanisms that regulate hematopoietic cell expansion in vitro by identifying defined culture conditions. We report the results of experiments with CD34(+) cells from cord blood (CB, n = 13), bone marrow (BM, n = 4), and mobilized peripheral blood stem cells (PBSC, n = 5) using two combinations of cytokines: (A) granulocyte colony-stimulating factor (G-CSF), interleukin-3 (IL-3), interleukin-6 (IL-6), stem cell factor (SCF), erythropoietin (EPO), insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (FGF-b) and (B) combination A plus FLT3 ligand (FL) and megakaryocyte growth and development factor (PEG rhMGDF). Cultures of immunoselected CD34(+) cells were performed in serum-free liquid medium without serum substitutes. The area under the curve (AUC) obtained by plotting the logarithm of the total number of viable cells, CD34(+) cells, and CFC per well, toward the week of culture was used as an index of cell expansion. With CB, a significant difference was obtained between the two combinations of cytokines with regard to the total number of viable cells, GM-CFC, and CD34(+) cells. The difference between the two combinations of cytokines obtained with BM was significant with respect to the total number of viable cells and CD34(+) cells but not for the erythroid and myeloid progenitors. When CD34(+) cells from peripheral blood stem cells (PBSC) were cultured in presence of the two combinations of cytokines, the difference in terms of AUC was not statistically significant. Our data indicate additional effects in terms of proliferation and expansion of hematopoietic cells in serum-free conditions when FL and polyethylene glycol (PEG) rhMGDF are included in culture and suggest a differential activity of these cytokines on cells from different hematopoietic sources.
本研究的目的是通过确定特定的培养条件来阐明体外调节造血细胞扩增的机制。我们报告了使用两种细胞因子组合对来自脐带血（CB，n = 13）、骨髓（BM，n = 4）和动员的外周血干细胞（PBSC，n = 5）的CD34(+)细胞进行实验的结果：（A）粒细胞集落刺激因子（G-CSF）、白细胞介素-3（IL-3）、白细胞介素-6（IL-6）、干细胞因子（SCF）、促红细胞生成素（EPO）、胰岛素样生长因子-1（IGF-1）、碱性成纤维细胞生长因子（FGF-b），以及（B）组合A加FLT3配体（FL）和巨核细胞生长与发育因子（聚乙二醇化重组人巨核细胞生长发育因子，PEG rhMGDF）。免疫选择的CD34(+)细胞培养在无血清液体培养基中，不添加血清替代物。通过绘制每孔活细胞、CD34(+)细胞和集落形成细胞（CFC）总数的对数相对于培养周数的曲线得到的曲线下面积（AUC）用作细胞扩增的指标。对于脐带血，两种细胞因子组合在活细胞总数、粒-巨噬细胞集落形成细胞（GM-CFC）和CD34(+)细胞方面存在显著差异。对于骨髓，两种细胞因子组合在活细胞总数和CD34(+)细胞方面存在显著差异，但在红系和髓系祖细胞方面无显著差异。当外周血干细胞（PBSC）的CD34(+)细胞在两种细胞因子组合存在的情况下进行培养时，AUC方面的差异无统计学意义。我们的数据表明，在无血清条件下培养时，当培养中包含FL和聚乙二醇（PEG）重组人巨核细胞生长发育因子（rhMGDF）时，造血细胞在增殖和扩增方面有额外的效应，并提示这些细胞因子对来自不同造血来源的细胞具有不同的活性。
