Evaluation of ex vivo expansion potential of cord blood and bone marrow hematopoietic progenitor cells using cell tracking and limiting dilution analysis.
Evaluation of ex vivo expansion potential of cord blood and bone marrow hematopoietic progenitor cells using cell tracking and limiting dilution analysis.
复制标题
DOI：
10.1182/blood.v85.8.2059.bloodjournal8582059
复制
发表时间：
1995-04
期刊：
影响因子：
20.3
通讯作者：
C. Traycoff;Steven T. Kosak;S. Grigsby;E. Srour
中科院分区：
文献类型：
--
作者：
C. Traycoff;Steven T. Kosak;S. Grigsby;E. Srour
在没有能够确定离体扩增的人造血祖细胞的功能性的结论性测定的情况下，我们结合细胞跟踪与膜染料PKH 2，CD 34的免疫染色，和有限稀释分析，以估计在新生的CD 34+细胞中的长期造血培养起始细胞（LTHC-IC）的频率。在第0天用PKH 2染色脐带血（CB）和骨髓（BM）CD 34+细胞，并在短期无基质细胞悬浮培养物中与干细胞因子（SCF）和白细胞介素-3（IL-3）一起培养。通过相对于第0天的PKH 2荧光和CD 34的持续表达追踪培养物中CD 34+细胞的增殖。因此，可以鉴定在维持CD 34表达的同时分裂的细胞（CD 34 + PKH 2dim）和表达CD 34但未分裂的其他细胞（CD 34 + PKH 2bright）。在所有这些培养物中，BM和CB CD 34+细胞的一部分不能响应于细胞因子而分裂，并且作为CD 34 + PKH 2bright细胞在培养物中持续长达10天。在培养的第5天和第7天之间，以有限稀释方案将CD 34 + PKH 2bright和CD 34 + PKH 2dim细胞分选到用培养基、SCF、IL-3、IL-6、粒细胞-巨噬细胞集落刺激因子和促红细胞生成素制备的96孔板中。2周后测定在单个威尔斯孔中增殖的细胞的克隆原祖细胞含量，并使用阴性威尔斯孔的百分比计算每个群体中LTHC-IC的频率。在新鲜分离的BM和CB CD 34+细胞中，LTHC-IC的频率分别为2.01% +/- 0.98%（平均值+/- SEM）和7.56% +/-2.48%。在培养5至7天后，3.00% +/-0.56%的离体扩增的BM CD 34 + PKH 2bright细胞和4.46% +/-1.10%的CD 34 + PKH 2dim细胞是LTHC-IC。相比之下，在离体扩增的CB CD 34+细胞中LTHC-IC的频率急剧下降，使得在培养5至7天后仅3.87% +/-2.06%的PKH 2bright和2.29% +/-1.75%的PKH 2dim细胞被确定为起始细胞。然而，当结合计算培养物中CD 34+细胞数量的净变化时，与新鲜分离的细胞相比，BM和CB细胞中的LTHC-IC的总和下降，尽管两种组织之间的程度不同。（400字处截断摘要）
In the absence of conclusive assays capable of determining the functionality of ex vivo expanded human hematopoietic progenitor cells, we combined cell tracking with the membrane dye PKH2, immunostaining for CD34, and limiting dilution analysis to estimate the frequency of long-term hematopoietic culture-initiating cells (LTHC-ICs) among de novo-generated CD34+ cells. Umbilical cord blood (CB) and bone marrow (BM) CD34+ cells were stained with PKH2 on day 0 and cultured with stem cell factor (SCF) and interleukin-3 (IL-3) in short-term stromal cell-free suspension cultures. Proliferation of CD34+ cells in culture was tracked through their PKH2 fluorescence relative to day 0 and the continued expression of CD34. As such, it was possible to identify cells that had divided while maintaining the expression of CD34 (CD34+PKH2dim) and others that expressed CD34 but had not divided (CD34+PKH2bright). In all such cultures, a fraction of both BM and CB CD34+ cells failed to divide in response to cytokines and persisted in culture for up to 10 days as CD34+PKH2bright cells. Between days 5 and 7 of culture, CD34+PKH2bright and CD34+PKH2dim cells were sorted in a limiting dilution scheme into 96-well plates prepared with medium, SCF, IL-3, IL-6, granulocyte-macrophage colony-stimulating factor, and erythropoietin. Cells proliferating in individual wells were assayed 2 weeks later for their content of clonogenic progenitors and the percentage of negative wells was used to calculate the frequency of LTHC-ICs in each population. Among fresh isolated BM and CB CD34+ cells, the frequencies of LTHC-ICs were 2.01% +/- 0.98% (mean +/- SEM) and 7.56% +/- 2.48%, respectively. After 5 to 7 days in culture, 3.00% +/- 0.56% of ex vivo-expanded BM CD34+PKH2bright cells and 4.46% +/- 1.10% of CD34+PKH2dim cells were LTHC-ICs. In contrast, the frequency of LTHC-IC in ex vivo expanded CB CD34+ cells declined drastically, such that only 3.87% +/- 2.06% of PKH2bright and 2.29% +/- 1.75% of PKH2dim cells were determined to be initiating cells after 5 to 7 days in culture. However, when combined with a calculation of the net change in the number of CD34+ cells in culture, the sum total of LTHC-ICs in both BM and CB cells declined in comparison to fresh isolated cells, albeit to a different degree between the two tissues.(ABSTRACT TRUNCATED AT 400 WORDS)