The proposed research will develop an in vitro differentiation system for effective production of functional neutrophils from human embryonic stem (ES) cells. These studies will be directed towards developing techniques for effective production of functional neutrophils from human ES cells to provide a model system for studying neutrophil differentiation and regulation in man, as well as for eventual therapeutic applications.
Specific aim #1 which contains four subsections will utilize four approaches to effectively produce neutrophils from human ES cells.
In specific aim 1 a) we will first evaluate if the ES/OP9 co-culture system developed by Lieber et al, 2003 for the in vitro differentiation of mouse ES cells into neutrophils can be used for production of neutrophils from human ES cells.
In specific aim #1 b), an assessment of the optimal embryoid body (EB) stage for production of neutrophil progenitors will be assessed.
Specific aim #1 c) will evaluate the importance of exogenously added gp-130 cytokines on neutrophil production from human ES cells.
In specific aim 1 d) we will evaluate the importance of feeder cells and feeder-derived M-CSF on the differentiation of human ES cells.
Specific aim #2 will focus on characterizing the ES cell-derived neutrophils with respect to their morphology, neutrophil markers and functional capabilities. Specifically, the expression of neutrophil associated antigens CD-14 and CD-15, CD-88, CD-114, as well as the neutrophil granule proteins lactoferrin and gelatinase will be assessed using flow cytometry and immunofluorescence microscopy. Neutrophils will also be evaluated for the presence of specific chloroacetate esterase which is a neutrophil marker. Assessment of the functional capacity of these neutrophils will include superoxide production in response to phorbol myrstal acetate (PMA), reduction of nitroblue tetrozolium for assessment of superoxide production on a single cell basis and their ability to undergo chemotaxis in response to IL-8 and KC. Finally, Specific Aim 3 will define the mechanisms by which human neutrophils are released from a stromal microenvironment during inflammation. ? ? ?
