Koizumi K, Sawada K, Sato N, Hirayama S, Yasukouchi T, Yamaguchi M, Takahashi T A, Sekiguchi S, Koike T
Department of Internal Medicine II, Hokkaido University School of Medicine, Sapporo, Japan.
Exp Hematol. 1994 Nov;22(12):1171-8.
We determined the appropriate incubation period to expand human peripheral blood (PB) CD34+ cells for clinical application and the role of recombinant human (rh) interleukin-3 (rhIL-3) in the expansion and differentiation of these cells. The cells were purified up to 40 +/- 16% and transitional changes in immunophenotypic subpopulations in suspension culture were examined following stimulation with three different combinations of rh colony-stimulating factors (rhCSFs): 1) rhIL-3 alone, 2) rhIL-6, rhSCF, rhG-CSF, plus rhGM-CSF, and 3) the four CSFs plus rhIL-3. With all three CSF combinations, the total cells increased continuously after day 5 until day 14, and a combination of the five CSFs always gave rise to the highest number of total cells. Immunophenotypic analysis of the expanded cells showed that with all three CSF combinations CD34+ cells reached a maximal rate on day 5 and then decreased in an inverse correlation between the logarithm of CD34 positive rate and the duration of suspension culture. The maximum expansion of CD34+ cells and PB progenitor cells (PBPC) with rhIL-3 alone, the four CSFs, or the five CSFs was observed on day 5, 10, or 7, respectively. The combination of the five CSFs was identified as the most potent stimulus for expansion of PBPC and CD34+ cells, as it increased non-erythroid PBPC 89 +/- 69-fold, with a range of 24 to 204-fold on day 7. However, differences in the expansion rate of these cells on days 5, 7, and 10 were not statistically significant. The majority of purified CD34+ cells coexpressed CD38 (91 +/- 3%) but were negative for CD33 (85 +/- 4%), and one-half coexpressed CD13. With all three combinations of CSFs, the mature CD34+ cells that coexpressed CD38, CD33, or CD13 expanded in parallel with the total CD34+ cells, while an increase in relatively immature CD34+ cells, which do not express CD38, CD33, or CD13, was only statistically significant with the five CSFs. Thus, rhIL-3 played a critical role when combined with the four CSFs by increasing both mature and immature CD34+ cells.
我们确定了用于临床应用的扩增人外周血（PB）CD34+细胞的合适培养期，以及重组人（rh）白细胞介素-3（rhIL-3）在这些细胞扩增和分化中的作用。细胞纯化至40±16%，并在三种不同组合的rh集落刺激因子（rhCSFs）刺激后，检测悬浮培养中免疫表型亚群的过渡变化：1）单独使用rhIL-3，2）rhIL-6、rhSCF、rhG-CSF加rhGM-CSF，3）四种CSF加rhIL-3。使用所有三种CSF组合时，总细胞数在第5天至第14天持续增加，五种CSF的组合总能产生最多的总细胞数。对扩增细胞的免疫表型分析表明，使用所有三种CSF组合时，CD34+细胞在第5天达到最大比例，然后在CD34阳性率的对数与悬浮培养持续时间之间呈负相关下降。单独使用rhIL-3、四种CSF或五种CSF时，CD34+细胞和PB祖细胞（PBPC）的最大扩增分别在第5天、第10天或第7天观察到。五种CSF的组合被确定为扩增PBPC和CD34+细胞的最有效刺激物，因为它使非红系PBPC增加了89±69倍，在第7天的范围为24至204倍。然而，这些细胞在第5天、第7天和第10天的扩增率差异无统计学意义。大多数纯化的CD34+细胞共表达CD38（91±3%），但CD33阴性（85±4%），一半共表达CD13。使用所有三种CSF组合时，共表达CD38、CD33或CD13的成熟CD34+细胞与总CD34+细胞平行扩增，而不表达CD38、CD33或CD13的相对不成熟CD34+细胞的增加仅在五种CSF组合时具有统计学意义。因此，rhIL-3与四种CSF联合使用时，通过增加成熟和不成熟的CD34+细胞发挥了关键作用。
