*PURPOSE: Infusions of donor hematopoietic stem cells (DHSCs) are routinely used in cancer therapy and are now being tested for tolerance induction in solid organ transplant recipients. However, the fate of infused DHSCs and the mechanism by which they contribute to tolerogenic states is not fully characterized. To better elucidate this, we studied the effect of allogeneic stimulation on HSC phenotype and function _in vitro. _ *METHODS: CD34+ HSCs were isolated from deceased donor bone marrow and stimulated with either irradiated, autologous spleen cells or allogeneic PBMCs in culture media supplemented with IL-2 and 50% MLR supernatant. DHSC cultures (DHSC-L) were phenotypically characterized by flow cytometry, and the immunoregulatory function was assessed in a series of MLR suppression assays. The cytotoxic capability of DHSC-L was analyzed using a standard chromium release assay. Newly generated T-cell receptor excision circles (Trecs) were quantified using real time PCR. *RESULTS: DHSCs proliferated and differentiated following allogeneic stimulation. Proportions of CD34+ cells decreased throughout the culture, with increased numbers of newly generated CD33+ myeloid cells and CD3+ T cells, including CD4+ and CD8+ subsets (Figure 1A) and CD4+CD127-CD25highFOXP3+Tregs (Figure 1B). Molecular analyses for Trecs demonstrated cells underwent recent TcR rearrangements. When tested as modulators in MLRs, DHSC-L and their purified subsets (CD33+, CD4+, CD8+, Lin-) exhibited significant immunoregulatory effects in a non-specific fashion (Figure 2 A & B). However, the immunosuppressive function of the CD8+ subset was mediated via direct cell lysis of responder cells (Figure 2C). *CONCLUSIONS: In this study, we have developed a potential method for _ex-vivo_ DHSC expansion into immunoregulatory cell lines that may be utilized for tolerance induction. These studies have also indicated that the thymic environment is not essential for T-cell generation. Extrapolation of these results to a clinical setting would suggest that DHSC infusion into transplant recipients would generate multiple subsets of donor “chimeric” cells that are immunoregulatory. [

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]CITATION INFORMATION: Sanders J., Leventhal J., Mathew J. In Vitro Differentiation of Hematopoietic Stem Cells Into Immunoregulatory T and Myeloid Cells Following Allogeneic Stimulation AJT, Volume 23, Issue 6, Supplement 1.

Jes Sanders MD

Northwestern University

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