Podestà M, Piaggio G, Pitto A, Zocchi E, Soracco M, Frassoni F, Luchetti S, Painelli E, Bacigalupo A
Divisione di Ematologia 2, Ospedale S. Martino, Genova, Italy.
Exp Hematol. 2001 Mar;29(3):309-14. doi: 10.1016/s0301-472x(00)00678-0.
The aim of this study was to compare the in vitro growth of cord blood-derived progenitors with that of bone marrow and peripheral blood.
We analyzed 192 umbilical cord blood (UCB), 35 normal bone marrow (NBM), and 35 granulocyte colony-stimulating factor (G-CSF)-primed normal peripheral blood (NPB) samples. Standard clonogenic assays (colony-forming unit granulocyte-macrophage [CFU-GM], burst-forming unit erythroid [BFU-E], CFU-granulocyte erythroid megakaryocyte macrophage [GEMM]) and standard long-term culture-initiating cell (LTC-IC) assay were performed. LTC-IC frequency also was tested under modified culture conditions. The variables tested were incubation temperature (37 degrees C and 33 degrees C) and supportive stromal cell lines (NIH3T3 and M210-B4).
The CFU-GM and CFU-GEMM frequencies of UCB samples were similar to NPB and higher compared to NBM samples (p < 10(-4) and p < 0.007 respectively). On the other hand, the BFU-E frequency was lower in cord blood samples (5.2 +/- 5.6/10(4) MNC) compared to bone marrow (7 +/- 3.8/10(4) MNC; p < 0.005) and peripheral blood (15.2 +/- 11.1/10(4) MNC; p < 10(-4)). All colony types (CFU-GM, BFU-E, CFU-GEMM) generated from cord blood progenitors were larger with respect to the other tissues. The LTC-IC frequency was markedly decreased (8.8 +/- 3.8/10(6) MNC) in cord blood with respect to bone marrow (40.7 +/- 7.4/10(6) MNC; p < 10(-4)) and peripheral blood (28.8 +/- 3.8/10(6) MNC; p < 0.04). However, when culture conditions (temperature, stromal layers) were modified, UCB-LTC-IC frequency significantly increased, while the growth of early progenitors derived from adult tissues (BM and PB) did not show any variation. Whatever culture conditions were used, the proliferative potential of UCB LTC-IC was significantly higher with respect to bone marrow and G-CSF-primed PB (10.6 +/- 7.7 colonies vs. 5.9 +/- 5 vs 3.2 +/- 2.2 colonies; p < 0.02 and p < 0.001 respectively).
Optimal conditions for estimation of the LTC-IC frequency in cord blood samples seem to be different from those usually applied to PB and BM progenitors. Although UCB hemopoietic progenitors have a higher proliferative potential than those from bone marrow and G-CSF-primed peripheral blood, their quantitation depends on the culture conditions, which makes it difficult to establish their exact number. This problem and the fact that a significant proportion of UCB samples grew poorly in culture make it necessary to develop suitable and standardized functional assays to test UCB progenitor content before the transplantation procedure.
本研究旨在比较脐血来源祖细胞与骨髓及外周血来源祖细胞的体外生长情况。
我们分析了192份脐带血（UCB）、35份正常骨髓（NBM）和35份经粒细胞集落刺激因子（G-CSF）动员的正常外周血（NPB）样本。进行了标准的集落形成试验（粒细胞-巨噬细胞集落形成单位[CFU-GM]、红系爆式集落形成单位[BFU-E]、粒细胞-红系-巨核细胞-巨噬细胞集落形成单位[GEMM]）以及标准的长期培养起始细胞（LTC-IC）试验。还在改良培养条件下检测了LTC-IC频率。所检测的变量包括孵育温度（37℃和33℃）以及支持性基质细胞系（NIH3T3和M210-B4）。
脐血样本的CFU-GM和CFU-GEMM频率与外周血相似，且高于骨髓样本（分别为p < 10⁻⁴和p < 0.007）。另一方面，脐血样本中的BFU-E频率低于骨髓（7 ± 3.8/10⁴单个核细胞；p < 0.005）和外周血（15.2 ± 11.1/10⁴单个核细胞；p < 10⁻⁴）（5.2 ± 5.6/10⁴单个核细胞）。脐血祖细胞产生的所有集落类型（CFU-GM、BFU-E、CFU-GEMM）相对于其他组织都更大。脐血中的LTC-IC频率相对于骨髓（40.7 ± 7.4/10⁶单个核细胞；p < 10⁻⁴）和外周血（28.8 ± 3.8/10⁶单个核细胞；p < 0.04）显著降低（8.8 ± 3.8/10⁶单个核细胞）。然而，当培养条件（温度、基质层）改变时，脐血LTC-IC频率显著增加，而成人组织（骨髓和外周血）来源的早期祖细胞生长未显示任何变化。无论使用何种培养条件，脐血LTC-IC的增殖潜能相对于骨髓和经G-CSF动员的外周血都显著更高（10.6 ± 7.7个集落对5.9 ± 5个集落对3.2 ± 2.2个集落；分别为p < 0.02和p < 0.001）。
估计脐血样本中LTC-IC频率的最佳条件似乎与通常应用于外周血和骨髓祖细胞的条件不同。尽管脐血造血祖细胞比骨髓和经G-CSF动员的外周血造血祖细胞具有更高的增殖潜能，但其定量取决于培养条件，这使得确定其确切数量变得困难。这个问题以及相当一部分脐血样本在培养中生长不佳的事实，使得有必要开发合适且标准化的功能试验，以便在移植程序前检测脐血祖细胞含量。
