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Cytomatrix |
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T Cell Generation in a Thymic Organoid | ||
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Thomas Meyer, Julie Reitter, Mark J Pykett, Richard Evans,
Mark Poznansky, Jerome Ritz, David T Scadden and Michael Rosenzweig. Cytomatrix, Woburn, MA, 01801, United States Experimental Hematology, Harvard-MGH, Boston, MA, 02129, United States; and Adult Oncology, Dana Farber Cancer Institute, Boston, MA, 02115, United States. |
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The in vitro generation of T lymphocytes has demonstrated an absolute dependency on stromal elements in the context of a three dimensional environment. We have previously described a culture system that incorporates an inert, biocompatible, three dimensional matrix (CellfoamTM), that when pre-established with murine thymic stroma, supports the in vitro differentiation of human Hematopoietic progenitors into mature T cells. These cells demonstrate a phenotype consistent with thymic differentiation as well as specific characteristics of de-novo T cell generation, such as an increase in the number of T cell receptor excisional circles (TRECs). In an effort to develop a clinically useful system, we have explored alternative sources of human stroma. We have shown that human skin stroma pre-established on the CellfoamTM matrix is also capable of supporting T cell differentiation. Skin tissue from adults was cultured in CellfoamTM for 2-3 weeks until a confluent stromal cell network was established. Freshly isolated human bone marrow AC133+ cells (depleted for CD2+ and CD3+ cells) were then co-cultured on the the stroma in CellfoamTM (about 2 weeks). Flow cytometry revealed that those non-adherent cells included CD4+/8- and CD4-/8+ single positive as well as some CD4+/8+ double positive T cells. These cells were mostly naïve T cells (CD45RA+) and expressed almost exclusively the TCR Bheterodimer. Using PCR the appearance of TRECs was detected indicating T cell receptor rearrangement occurred during T cell maturation. Spectratype and analysis demonstrated a diverse T cell repertoire. By means of both phenotypic and functional evaluation of these cells, we have demonstrated that human T cells can be effectively generated from bone marrow progenitor cells in the context of a three dimensional microenvironment. The use of alternative sources of stroma provides the possibility that autologous, readily accessible sources of tissue may be used. Furthermore, the system presents the potential for exploring human T-cell selection processes and manipulating them for ex vivo generation of antigen-reactive cells.   |
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