Cytokine Receptor Expression on Hematopoietic Progenitor Cells is the research focus of the Laboratory of Flow Cytometry. Hematopoietic cells are derived from a small pool of multipotential stem cells, which differentiate into committed progenitor cells for each hematopoietic lineage.  These cells further expand to produce functional end cells.  Antibodies have been produced to many of the proteins on the membrane of hematopoietic cells that include cytokine receptors, adhesion molecules, activation molecules and metabolic molecules.  Since a unique repertoire of these proteins is displayed for each subset along the differentiation pathway, we are able to separate and identify specific cell populations using Flow Cytometry. 

Hematopoietic cells are responsive to a number of cytokines.  We hypothesize that hematopoietic cell subsets express a unique repertoire of cytokine receptors whose engagement by ligand regulates both proliferation and differentiation.  This regulation can be directed by altering the combination of cytokines within the cell’s immediate microenvironment.  If this hypothesis is true, all members of any hematopoietic lineage could be represented in culture once provided the correct cytokine cocktail.  Thus, our research is focused on determining the correct cytokine stem cell cocktail.  To accomplish this we have developed a process to simultaneously immunophenotype cells and to evaluate their cytokine receptor and ligand expression.  Immunophenotyping is the term applied to describe the process of determining specific proteins on the membrane or inside a cell.  Because we can measure up to seven colors of fluorescence, we can use antibodies to identify cells, their cytokine receptors and their intracellular cytokines simultaneously. This is important because coexpression can be objectively made and properly assigned.  Using this strategy, the growth factor repertoire for each lineage will be determined so the correct cocktail of growth factors can be assigned to a specific hematopoietic lineage.  Growth and differentiation can then be measured in culture.  Subsets can be sorted to provide highly purified committed precursor cells to insure lineage purity and fidelity. 

In addition, bystander or stromal cells may be important to the endogenous production of cytokines.  The cytokines that accumulate in culture are currently not known and may be most important for modulating a response.  Using a new flow cytometry based immunoassay procedure; the endogenous production of cytokines will also be determined.  Briefly, this assay uses colored microspheres to which a capture antibody for a cytokine is bound.  Since each bead set has a different color that can be resolved by the flow cytometer, multiple analytes can be measured in the same sample tube.  By using reporter antibodies, all with the same fluorochrome, the concentration of each analyte is determined.  The system is currently able to measure 64 separate analytes in a single specimen.  Our initial studies are focused on the production of dendritic cells and NK cells.  This is because significant gains have already been made to our understanding of their growth factor and differentiation requirements.  It is expected that this systematic approach to defining the needs of hematopoietic cells will provide an understanding of the regulation requirements of hematopoiesis.