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

首先，我们调查了骨髓增生异常综合征（MDS：24例）、再生障碍性贫血（AA：5例）和阵发性睡眠性血红蛋白尿症（PNH：3例）患者骨髓（BM）单个核细胞（MNC）中侧群（SP）细胞的频率。MDS、AA、PNH和健康骨髓中SP细胞群的平均值分别为0.62%、0.006%、0.42%和0.23%。MDS、PNH和健康BM之间的这些频率无显著性差异。按WHO分型，SP细胞检出率分别为RA 0 ~ 1.41%，RARS 0.03- 0.57%，RCMD 0.11- 2.04%，RAEB 1 1.18- 2.63%，RAEB 2 3.11%（1例）。接下来，为了确认MDS样品中SP细胞的克隆性，我们通过FACS细胞分选仪从两个8三体病例和一个7单体病例中分选SP细胞，并通过荧光原位杂交（FISH）研究其在原始样品中观察到的复发性染色体异常。在这三种情况下，SP细胞数量均高于正常对照组。 ...更多信息 涉及异常克隆。同时，检测ABCG 2蛋白在BMMNC中的表达。MDS、AA和PNH的BMMNC中ABCG 2+细胞的平均值分别为3.73%、0.01%和1.99%。流式细胞术分析ABCG 2表达结果较SP细胞法稳定，且各样本间差异较小。流式细胞仪多标记分析表明，MDS和PNH的SP细胞和ABCG 2+细胞群同时表达CD 34或CD 133，而不表达更高分化造血细胞的表面标记（CD 19、CD 3和CD 33）。因此，为了研究SP细胞和ABCG 2+细胞作为造血干细胞（HSC）的特性，我们对这些群体应用长期培养起始细胞（LTC-IC）测定和鹅卵石区域形成细胞（CAFC）测定。AA患者的LTC-IC和CAFC均明显降低。MDS和PNH患者中LTC-IC和CAFC的频率低于正常骨髓。此外，在MDS病例中，形成每个CA的细胞数少于健康BM中的细胞数，并且在相同的生长因子刺激条件下，MDS样品中的CA比健康BM样品（5-7周）更早（3-5周）消退。总之，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