Functional and phenotypic characterization of cord blood and bone marrow subsets expressing FLT3 (CD135) receptor tyrosine kinase.

The class III receptor tyrosine kinase FLT3/FLK2 (FLT3; CD135) represents an important molecule involved in early steps of hematopoiesis. Here we compare cell-surface expression of FLT3 on bone marrow (BM) and cord blood (CB) cells using monoclonal antibodies (MoAbs) specific for the extracellular domain of human FLT3. Flow cytometric analysis of MACS-purified BM and CB cells showed that 63% to 82% of BM CD34+ and 88% to 95% of the CB CD34+ cells coexpress FLT3.

Specificity and functional effects of antibodies to human stem cell factor.

Three monoclonal antibodies (Mabs), 7H6, 4B10 and Genzyme Mab, and a commercially-available polyclonal antiserum (Genzyme) to human Stem Cell Factor (SCF) were compared for their ability to detect native and recombinant SCF in a variety of assays, and for blocking of SCF function. All antibodies were found to bind to the membrane bound isoform as well as soluble SCF and to bind to both glycosylated (yeast MGF) and unglycosylated (E. coli SCF) recombinant factor.

A powerful new technique for isolating genes encoding cell surface antigens using retroviral expression cloning.

cDNA expression cloning using retroviral vectors provides a means of stably introducing genes into target cells at efficiencies that surpass those achieved by transfection. Furthermore, retroviral vectors allow for the introduction and expression of complex cDNA libraries in a wide range of cell types, including cells of hemopoietic origin. Here we report a novel method for rapidly isolating genes encoding cell surface molecules (CSM) from a human bone marrow stromal cell cDNA library constructed in the retroviral vector, pRUFneo.

Primitive human hematopoietic progenitors adhere to P-selectin (CD62P).

P-selectin was shown to bind committed human hematopoietic progenitors (colony-forming unit-granulocyte-macrophage [CFU-GM] and burst-forming unit-erythroid [BFU-E]) as identified by their expression of the CD34 antigen and by in vitro clonogenic assays. In addition, P-selectin bound all precursors (pre-CFU) of committed myeloid progenitors assayed by their ability to sustain hematopoiesis in both conventional stroma-containing and stroma-free, cytokine-dependent systems. Binding of CD34+ cells to P-selectin was temperature-independent and shear-resistant, occurred only in the presence of divalent cations, was protease sensitive, and was completely blocked by anti-P-selectin antibody.

Potential adhesion mechanisms for localisation of haemopoietic progenitors to bone marrow stroma.

Haemopoiesis occurs in intimate physical association with the stromal elements of the bone marrow. Current evidence supports the hypothesis that the restriction of primitive haemopoietic progenitor cells (HPC) to the bone marrow involves developmentally regulated adhesive interactions between HPC and the stromal cell microenvironment. This review examines the expression and function of cell adhesion molecules (CAM) on human HPC and marrow stromal cells.

Isolation, characterization and functional activity of human marrow stromal progenitors in hemopoiesis.

In conclusion, monoclonal antibody STRO-1 has proven to be an extremely valuable reagent for the identification, isolation and functional characterization of human bone marrow stromal cell precursors. We were able to isolate CFU-F free of contaminating hemopoietic progenitors and are beginning to construct a detailed picture of their cell surface phenotype and have developed assays to examine the conditions required for their growth and differentiation. In the future, we hope to determine whether multipotential stromal stem cells exist in human bone marrow and to establish culture conditions that selectively promote self-renewal or development along any of a number of stromal cell lineages.