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Phenotype analysis of hematopoietic CD34+ cell populations derived from
human umbilical cord blood using flow cytometry and cDNA-polymerase chain
reaction
SJ Thoma, CP Lamping and BL Ziegler
Department of Clinical Physiology & Occupational Medicine, University
of Ulm, Germany.
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.
Volume 83,
Issue 8,
pp. 2103-2114,
04/15/1994
Copyright © 1994 by The American Society of Hematology
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