Becton Dickinson and Company Limited

Peter Thompson

Fridtjof Lund-Johansen

Johanna Olweus

Fridjtof Lund-Johannsen

The present invention demonstrates that M-CSF responsiveness and the M-CSFR expression can be used to discriminate monocytic and granulocytic cells within a population of cells which strongly expresses the CD34 antigen (CD34.sup.hi). Briefly, the method comprises isolating phenotypically and functionally defined CD34.sup.+ subsets, and staining with anti-M-CSFR monoclonal antibodies to measure expression on these primitive progenitors and cells committed to the granulocytic and monocytic lineages, based upon expression of M-CSFR. CD34.sup.hi M-CSFR.sup.hi cells are highly clonogenic and approximately 70% of the colonies are CFU-M (monocytic), whereas less than 20% were CFU-G (granulocytic). In contrast, CD34.sup.hi cells that were positive for the granulo-monocytic marker CD64 and negative for the M-CSFR contained high frequencies of 91% pure CFU-Gs. After 60 h in culture, CD34.sup.hi M-CSFR.sup.hi cells developed into distinct populations of M-CSFR.sup.hi and M-CSFR.sup.lo cells. These two populations gave rise almost exclusively to monocytes and granulocytes, respectively. This result demonstrates that M-CSF target specificity among human hematopoietic progenitor cells is determined by lineage-specific regulation of the M-CSFR and demonstrate that M-CSFR is a useful marker to discriminate CFU-Ms from CFU-Gs.

Differentiation of granulocytic and monocytic progenitor cells

A novel approach to study changes in protein tyrosine phosphorylation during apoptosis, and thereby identify cells committed to apoptosis is presented, methods used to study apoptosis and tyrosine phosphorylation at the single cell level are combined to study directly whether apoptosis in hematopoietic cells is associated with changes in cellular phosphotyrosine levels. The changes in cellular phosphotyrosine content strongly correlated with the appearance of features of cell death such as cell shrinkage, DNA fragmentation and loss of membrane integrity.

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Danville, CA, United States of America

Peter Thompson