Characterization of side population cells in bone marrow of myelodysplastic syndrome, aplastic anemia, and paroxysmal nocturnal hemoglobinuria.

首先，我们研究了骨髓增生异常综合征（MDS: 24例）、再生障碍性贫血（AA: 5例）和阵发性夜间血红蛋白尿（PNH: 3例）患者骨髓（BM）单核细胞（MNC）侧群（SP）细胞的频率。MDS、AA、PNH和健康BM的SP细胞群平均值分别为0.62%、0.006%、0.42%和0.23%。这些频率在MDS、PNH和健康BM之间没有显著性差异。对于MDS的WHO亚型，我们可以检测到SP细胞的频率如下：RA 0 ~ 1.41%, RARS 0.03 ~ 0.57%, RCMD 0.11 ~ 2.04%, RAEB1 1.18 ~ 2.63%, RAEB2 3.11%（1例）。接下来，为了确认MDS样本中SP细胞的克隆性，我们利用FACS细胞分选器对2例8三体和1例7单体的SP细胞进行了分选，并利用荧光原位杂交（FISH）对其在原始样本中观察到的复发性染色体异常进行了研究。在这三种情况下，都表明SP细胞的增殖与异常克隆有关。同时检测ABCG2蛋白在BMMNC中的表达。MDS、AA和PNH患者BMMNC中ABCG2+细胞的平均值分别为3.73%、0.01%和1.99%。与SP细胞法相比，FACS法测定的ABCG2表达结果更稳定，且各样品和各检查结果差异较小。通过FACS多标记分析发现，MDS和PNH的SP细胞和ABCG2+细胞群体同时表达CD34或CD133，但未表达分化程度更高的造血细胞表面标记（CD19、CD3和CD33）。因此，为了研究SP细胞和ABCG2+细胞作为造血干细胞（HSC）的特性，我们对这些群体应用了长期培养启动细胞（LTC-IC）实验和鹅卵石区域形成细胞（CAFC）实验。AA组LTC-IC和CAFC均明显降低。在MDS和PNH病例中，LTC-IC和CAFC的频率低于健康BM。此外，在MDS病例中，形成每个CA的细胞数量少于健康BM，并且在相同的生长因子刺激条件下，MDS样本中的CA比健康BM样本（5-7周）更早（3-5周）回归。综上所述，通过对SP细胞和ABCG+细胞群的分析，表明MDS/AA/PNH中HSC的数量和功能异常。少

At first, we investigated frequency of side population (SP) cells in bone marrow (BM) mononuclear cells (MNC) of clinical cases with myelodysplastic syndrome (MDS : 24 cases), aplastic anemia (AA : 5 cases), and paroxysmal nocturnal hemoglobinuria (PNH : 3 cases). Mean values of SP cell population in MDS, AA, PNH, and healthy BM were 0.62%, 0.006%, 0.42%, and 0.23%, respectively. There was no significance in these frequencies between MDS, PNH, and healthy BM. With regard to WHO subtypes of MDS, we could detect the frequency of SP cells as follows ; RA 0-1.41%, RARS 0.03-0.57%, RCMD 0.11-2.04%, RAEB1 1.18-2.63%, and RAEB2 3.11% (one case). Next, to confirm clonality of SP cells in MDS samples, we sorted SP cells from two cases with trisomy 8 and one case of monosomy 7 by FACS cell sorter, and investigated their recurrent chromosomal abnormalities, which were observed in original samples, by fluorescence in situ hybridization (FISH). In all three cases, it was indicated that SP cell popu … More lation involved abnormal clones. Concurrently, expression of ABCG2 protein in BMMNC was tested. Mean values of ABCG2+ cells in BMMNC of MDS, AA, and PNH were 3.73%, 0.01%, and 1.99%, respectively. Results of ABCG2 expression analysis by FACS were more stable and indicated less difference in each sample and examination than those of SP cell assay. By multi-marker analysis by FACS, it was indicated that the population of SP cells and ABCG2+ cells of MDS and PNH expressed CD34 or CD133 simultaneously, but didn't expressed surface markers of more differentiated hematopopietic cells (CD19, CD3, and CD33). Therefore, to investigate properties of SP cells and ABCG2+ cells as hematopoietic stem cells (HSC), we applied long-term culture-initiated cells (LTC-IC) assay and cobblestone area forming cells (CAFC) assay to these populations. In AA cases, both LTC-IC and CAFC were markedly reduced. In MDS and PNH cases, the frequencies of LTC-IC and CAFC were lower than healthy BM. Furthermore, in MDS cases, cell numbers forming each CA were less than in healthy BM, and CAs in MDS samples were regressed earlier (in 3-5 weeks) than in healthy BM samples (5-7 weeks) by same stimulatory condition of growth factors. Taken together, quantitative and functional abnormalities of HSC in MDS/AA/PNH were indicated through the analysis of SP cell and ABCG+ cell populations. Less