In vitro generation of hematopoietic progenitors and functional T cells from pluripotent stem cells



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The use of both multipotent progenitors and fully differentiated cells has been demonstrated to be effective for cell-based immunotherapy. The goal of this thesis was to establish an in vitro hematopoietic differentiation system to generate hematopoietic progenitor cells (HPCs) and functional T cells from pluripotent stem cells. Generation of progenitor T cells by co-culturing stem cells on Notch ligand-expressing OP9 stromal cells (OP9-DL1) had been successfully employed previously. However, further differentiation of these cells in vitro into mature, antigen-specific, functional T cells, without retroviral transduction of T cell receptors (TcRs), had not been achieved. In the thymic niche, differentiation of T cells to a state of antigen specificity is controlled by the interaction of their developing TcRs with the Major Histocompatibility Complex (MHC) on thymic stromal cells. We hypothesized that, by providing exogenous antigen-specific MHC/TcR signals, stem and progenitor cells could be engineered into functional effector T cells specific for the same antigen. In Chapter 3 and 4, we demonstrate that both thymus-derived double positive (DP: CD4+CD8+) immature T cells and mouse Embryonic Stem (ES) cells can be efficiently differentiated into antigen-specific CD8+ T cells using either MHC tetramers or peptide-loaded stromal cells. DP cells, following MHC/TcR signaling, retained elevated RAG1 levels, suggesting continuing TcR gene rearrangement. Both DP and ES cell-derived CD8+ T cells showed significant Cytotoxic T Lymphocyte (CTL) activity against antigen-loaded target cells, indicating that these cells are functional. This directed differentiation strategy could provide an efficient method for generating functional, antigen-specific CTLs from stem cells for potential use in adoptive T cell therapies.
The use of ES cells in the clinic has been hindered by the unavailability of patient-specific ES cells and the ethical issues surrounding the use of human embryos. Induced pluripotent stem (iPS) cells offer great hope to regenerative medicine as their use can circumvent both the patient-specific and ethical issues associated with ES cells. In Chapter 5, we have developed a feeder cell-free suspension culture system supplemented with OP9-DL1 secretary factors to efficiently generated HPCs from iPS and ES cells. The differentiation potential of these HPCs was demonstrated by generation of DCs in the presence of GM-CSF and IL-3. The DCs express the activation molecules, CD86 and CD80 in response to LPS stimulation and are able to stimulate T cell proliferation in a mixed lymphocyte reaction. We employed extensive quantitative RT-PCR analysis to identify a number of differentially expressed genes in HPCs generated from the feeder-free culture.