Phenotype analysis of hematopoietic CD34+ cell populations derived from human umbilical cord blood using flow cytometry and cDNA-polymerase chain reaction

Abstract

A strategy to phenotype rare populations of hematopoietic cells expressing the cell-surface marker CD34 was studied. The antigenic phenotype of umbilical cord 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.