The monoclonal antibody W7C5 defines a novel surface antigen on hematopoietic stem cells.

We recently raised a monoclonal antibody, termed W7C5, against a surface antigen that is expressed at low levels on bone marrow and peripheral blood CD34+ stem/progenitor cells but at high levels on fetal liver CD34+ cells. A reasonable staining intensity was achieved using magnetofluorescent liposome conjugates to analyze expression of W7C5 antigen on CD34+CD38- bone marrow (BM) cells. Flow cytometric analyses revealed that W7C5 detects about 50% of immature CD34+CD38- BM cells that coexpressed the differentiation antigens CD164, CD133, and CD172a (SIRP alpha). In addition, W7C5 also recognized a CD34- BM fraction. These cells were negative for CD117 and CD133, but expressed CD45 and moderate levels of CD164. Injection of selected CD34+W7C5+ and CD34-W7C5+ cells into 55-60-day-old fetal sheep resulted in an engraftment of both fractions. Partial amino acid sequence analysis of affinity-purified lysates of KU-812 cells revealed that W7C5 detects a novel membrane protein. Together, W7C5 defines a novel molecule that is expressed on CD34+ as well as on CD34- stem cell subsets.

The basophil activation marker defined by antibody 97A6 is identical to the ectonucleotide pyrophosphatase/phosphodiesterase 3.

It has recently been shown that monoclonal antibody (MoAb) 97A6 detects a surface antigen expressed on basophils and their CD34(+) precursor cells, as well as the basophil cell line KU-812. In this report the partial amino acid sequence of affinity chromatography- and sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated 97A6 antigen(s) from KU-812 lysates was determined. Sequence alignment of high-performance liquid chromatography-selected tryptic peptides from the resulting 130- and 150-kd bands revealed a 100% identity with amino acids 393 to 405 of ectonucleotide pyrophosphatase/phosphodiesterase-3 (E-NPP3; CD203c) but not of the related ectoenzyme PC-1 (E-NPP1). Moreover, MoAb 97A6 selectively recognized 293 cells transfected with human E-NPP3, but did not react with cells transfected with PC-1 or parental 293 cells. In addition, E-NPP3 messenger RNA expression was detected in basophils but not other peripheral blood cells. Finally, MoAb 97A6 immunoprecipitated phosphodiesterase activity from KU-812 cells and peripheral blood basophils, but not from other cell populations. These data demonstrate that MoAb 97A6 recognizes the functionally active type II transmembrane ectoenzyme E-NPP3.

Unicellular-unilineage erythropoietic cultures: molecular analysis of regulatory gene expression at sibling cell level.

In vitro studies on hematopoietic control mechanisms have been hampered by the heterogeneity of the analyzed cell populations, ie, lack of lineage specificity and developmental stage homogeneity of progenitor/precursor cells growing in culture. We developed unicellular culture systems for unilineage differentiation of purified hematopoietic progenitor cells followed by daughter cell analysis at cellular and molecular level. In the culture system reported here, (1) the growth factor (GF) stimulus induces cord blood (CB) progenitor cells to proliferate and differentiate/mature exclusively along the erythroid lineage; (2) this erythropoietic wave is characterized by less than 4% apoptotic cells; (3) asymmetric divisions are virtually absent, ie, nonresponsive hematopoietic progenitors with no erythropoietic potential are forced into apoptosis; (4) the system is cell division controlled (cdc), ie, the number of divisions performed by each cell is monitored. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was applied to this culture system to investigate gene expression of diverse receptors, markers of differentiation, and transcription factors (EKLF, GATA-1, GATA-2, p45 NF-E2, PU.1, and SCL/Tal1) at discrete stages of erythropoietic development. Freshly isolated CD34(+) cells expressed CD34, c-kit, PU.1, and GATA-2 but did not express CD36, erythropoietin receptor (EpoR), SCL/Tal1, EKLF, NF-E2, GATA-1, or glyocophorin A (GPA). In early to intermediate stages of erythroid differentiation we monitored the induction of CD36, Tal1, EKLF, NF-E2, and GATA-1 that preceeded expression of EpoR. In late stages of erythroid maturation, GPA was upregulated, whereas CD34, c-kit, PU.1, and GATA-2 were barely or not detected. In addition, competitive single-cell RT-PCR was used to assay CD34 mRNA transcripts in sibling CD34(+)CD38(-) cells differentiating in unilineage erythroid cultures: this analysis allowed us to semiquantitate the gradual downmodulation of CD34 mRNA from progenitor cells through their differentiating erythroid progeny. It is concluded that this novel culture system, coupled with single-cell RT-PCR analysis, may eliminate the ambiguities intrinsic to molecular studies on heterogeneous populations of hematopoietic progenitors/precursors growing in culture, particularly in the initial stages of development.