This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. To advance human embryonic stem cells and their derivatives towards clinical application for treatment of blood disease. During last year our research was focused on development technique for high scale production of dendritic cells (DCs) from hES cells. We have established a system for directed differentiation of hES cells into myeloid DCs. As a first step, we induced hematopoietic differentiation by coculture of hES cells with OP9 stromal cells, and then expanded myeloid cells with GM- CSF using a feeder-free culture system. Myeloid cells had a CD4+CD11b+CD11c+ CD16+ CD123lowHLA-DR- phenotype, expressed myeloperoxidase, and included a population of M-CSFR+ monocyte-lineage committed cells. Further culture of myeloid cells in serum-free medium with GM-CSF and IL-4 generated cells that had typical dendritic morphology; expressed high levels of MHC class I and II molecules, CD1a, CD11c, CD80, CD86, DC-SIGN, and CD40; and were capable of antigen processing, triggering na ve T cells in MLR and presenting antigens to specific T cell clones through the MHC class I pathway. Incubation of DCs with A23187 calcium ionophore for 48 hours induced an expression of mature DC markers CD83 and fascin. The combination of GM-CSF with IL-4 provided the best conditions for DC differentiation. DCs obtained with GM-CSF and TNF-_ coexpressed a high level of CD14, and had low stimulatory capacity in MLR. These data clearly demonstrate that hES cells can be used as a novel and unique source of hematopoietic and DC precursors as well as DCs at different stages of maturation to address essential questions of DC development and biology. In addition, since ES cells can be expanded without limit, they can be seen as a potential scalable source of cells for DC vaccines or DC-mediated induction of immune tolerance. It is important to emphasize that using the described technique, we were able to grow up to 4x107 DCs from 107 hES cells initially cocultured with OP9 in six 10 cm tissue culture dishes; so already, a sufficient number of cells for functional studies and genetic manipulation can be generated.
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