Thymus, spleen and BM were removed and analysed by flow cytometry, PCR and functional assays. CellFoam cell-culture dishes 10 mm in diameter×1 mm in depth with an average pore density of 80 pores per inch (Cytomatrix) were pre-cultured
with fragmented thymic or skin tissue as described previously 13. After 22–35 days purified huCD34+ HSCs (1×105) were added onto the stroma-pre-cultured CellFoam matrices. Medium was changed every 3–4 days and non-adherent cells were harvested on day 14 (skin) or 21 (thymus). In some of the control experiments, fludarabine (GRY-Pharma) was additionally used at a concentration of 4 μg/mL prior to huCD34+ HSCs seeding. Expression levels of Notch-1 and its ligands RO4929097 mouse DLL-1 and -4 were analysed using standard procedures on an ABI 7300 (Applied Biosystems, Darmstadt, Germany). Primer sequences can be obtained from the corresponding author upon request. Supernatant cells from cell cultures or single-cell suspensions from spleen, thymus and BM of transplanted mice were analysed by flow cytometry (all CD markers obtained from BD) on a LSRII. Anti-HLA-B7 antibody was purchased from onelambda (BMT GmbH). The lineage cocktail, used to exclude committed haematopoietic precursors, contained CD3, CD14, CD15, CD19 and CD56 (all from BD). TCR repertoire diversity was analysed using standard CDR3-size fragment size analysis. After RT-PCR,
amplicons were detected on an ABI310 capillary sequencer and analysed with GeneMapper software (Applied Biosystems). Colony-forming capacity of stem cells was determined using a commercial CFC-assay (Stem Cell Technologies, containing SCF, JQ1 price GM-CSF, G-CSF, IL-3 and EPO). Briefly, 2×103 CD34+ or 2×104 CTLPs were cultured for 15 days in semi-solid medium and then analysed for the presence of colony-forming units of granulocytes/macrophages (CFU-GM) or erythrocytes (CFU/BFU-E) using an inverted
cell-culture microscope (Leica Microsystems, DM IRB, Wetzlar, Germany). Splenocytes were expanded with 100 U/mL IL-2 und 5 ng/mL IL-7 (Immunotools) for 10 days and then stimulated with PMA (50 ng/mL) and Ionomycin (750 ng/mL, Sigma) with addition of BrefeldinA (10 μg/mL) for the last hour before analysis. Production of IFN-γ and IL-4 was measured by intracellular flow cytometry using standard procedures. PRKACG For statistical comparison of results, we used the nonparametric Wilcoxon test for unpaired samples. A p-value of <0.05 was considered statistically significant. The authors thank Dr. Gerd Klein, University of Tübingen for providing aliquots of cDNA from isolated thymic epithelial cells for PCR analysis. Furthermore, the authors thank Mohammed Alkahled for his dedicated animal care. The authors thank the Merck KgaA company (Darmstadt, Germany) for kindly providing aliquots of the Fc-IL-7 fusion protein. H. Z. is the recipient of a scholarship from the Jürgen-Manchot Foundation. This work was supported by a grant from the Wilhelm-Sander-Foundation (♯2003.023.1, awarded to M. E. and K. S.
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