Thoma S J, Lamping C P, Ziegler B L
Department of Clinical Physiology & Occupational Medicine, University of Ulm, Germany.
Blood. 1994 Apr 15;83(8):2103-14.
A strategy to phenotype rare populations of hematopoietic cells expressing the cell-surface marker CD34 was studied. The antigenic phenotype of umbilical core blood (CB) CD34+ cells was investigated using flow cytometry and compared with the mRNA-phenotype determined by cDNA-polymerase chain reaction (cDNA-PCR) analysis. The cDNA-PCR method allowed an mRNA evaluation of small numbers of cells. Monoclonal antibodies and oligonucleotide primers that recognize myeloid, lymphoid, erythroid and platelet/megakaryocytic cell membrane antigens or corresponding mRNA transcripts were used. Evaluation by flow cytometry showed that the vast majority of CD34+ CB cells coexpressed CD38, CD18, HLA-DR, and CD33. Rare subpopulations of CD34+CD38-, CD34+CD18-, CD34+HLA-DR-, and CD34+CD33- were also identified. A large proportion of CD34+ CB cells expressed CD13, CD45R, and to a lesser extent CD71. The CD36, CD51, and CD61 antigens were identified on a small number of CD34+ cells. The three-color flow cytometry analysis showed that CD34+ cells stained with antibodies to CD61 and CD36 or CD51 can be divided into subsets that may represent progenitor cells committed to the erythroid and/or megakaryocytic lineage. A variety of other lineage-specific cell-surface antigens including pre-T-cell marker CD7 and markers of early B cells, ie, CD10 and CD19, were not coexpressed with CD34+. Using the cDNA-PCR it was seen that the mRNA phenotype of a small number of sorted CD34+ cells (purity > 98%) was negative for the markers CD2, CD14, CD16, CD20, CD21, CD22, CD41b, and glycophorin A that are expressed on differentiated cells but positive for CD34, CD7, CD19, CD36, and CD61. The results suggest that circulating CD34+CD7+ and CD34+CD19+ CB cells cannot be distinguished by flow cytometry but can be detected by cDNA-PCR. This indicates that CB either contains very low numbers of these progenitors or that the antigen density of CD7 and CD19 on CD34+ cells is below the detection limit of the flow cytometer. In contrast to flow cytometry, cDNA-PCR allows the phenotypic analysis of cells even if their number is small. Thus, the cDNA-PCR method can be useful in linking phenotype analyses, ie, markers of differentiation, to studies on gene expression within rare populations of hematopoietic stem cells.
研究了一种对表达细胞表面标志物CD34的造血细胞稀有群体进行表型分析的策略。使用流式细胞术研究了脐血（CB）CD34+细胞的抗原表型，并与通过cDNA聚合酶链反应（cDNA-PCR）分析确定的mRNA表型进行了比较。cDNA-PCR方法能够对少量细胞进行mRNA评估。使用了识别髓系、淋巴系、红系和血小板/巨核细胞细胞膜抗原或相应mRNA转录本的单克隆抗体和寡核苷酸引物。流式细胞术评估显示，绝大多数CD34+ CB细胞共表达CD38、CD18、HLA-DR和CD33。还鉴定出了CD34+CD38-、CD34+CD18-、CD34+HLA-DR-和CD34+CD33-的稀有亚群。很大比例的CD34+ CB细胞表达CD13、CD45R，且在较小程度上表达CD71。在少数CD34+细胞上鉴定出了CD36、CD51和CD61抗原。三色流式细胞术分析表明，用抗CD61和CD36或CD51抗体染色的CD34+细胞可分为可能代表定向于红系和/或巨核细胞系祖细胞的亚群。包括前T细胞标志物CD7和早期B细胞标志物（即CD10和CD19）在内的多种其他谱系特异性细胞表面抗原不与CD34+共表达。使用cDNA-PCR发现，少量分选的CD34+细胞（纯度>98%）的mRNA表型对于在分化细胞上表达的标志物CD2、CD14、CD16、CD20、CD21、CD22、CD41b和血型糖蛋白A呈阴性，但对于CD34、CD7、CD19、CD36和CD61呈阳性。结果表明，循环中的CD34+CD7+和CD34+CD19+ CB细胞无法通过流式细胞术区分，但可通过cDNA-PCR检测到。这表明脐血中要么含有极少量的这些祖细胞，要么CD34+细胞上CD7和CD19的抗原密度低于流式细胞仪的检测限。与流式细胞术不同，即使细胞数量很少，cDNA-PCR也能对细胞进行表型分析。因此，cDNA-PCR方法可用于将表型分析（即分化标志物）与造血干细胞稀有群体内的基因表达研究联系起来。
