Bone marrow CD34+ cells generate fewer T cells in vitro with increasing age and following chemotherapy

To assess the influence of high-dose chemotherapy and age on the intrinsic capacity of stem cells to generate T cells, CD34(+) cells derived from bone marrow used in clinical transplantation were evaluated in an in vitro T-ce ll assay using a mouse thymic microenvironment. CD34(+) cells were sorted f rom healthy donor and autologous back-up bone marrow after density gradient centrifugation and depletion for CD1, 3, 4, 7, 8, 19 and glycophorin A usi ng magnetic beads. CD34(+) cells were then introduced in day 14-15 fetal SC ID mouse thymus lobes by incubation in hanging drops for 48 h. After transf er to gelfoam rafts they were cultured for variable time periods, The robes were then homogenized in a tissue grinder for now cytometric analysis gati ng on human cells. These were evaluated for CD4, CD8, CD3 and HLA-DR surfac e expression 51 samples were analysed and three patterns of T-cell precurso r development could be observed. In pattern A no human cells could be recov ered, in pattern B maturation stopped at the CD4(+)CD8(-)CD3(-) pre-T-cell stage, and in pattern C maturation to double-positive CD4(+)CD8(+) thymocyt es was reached. In 25 healthy donors under age 40 three showed pattern A, 1 2 pattern B and 10 pattern C, whereas in 16 healthy donors over the age 40 there were respectively four with A, seven with B and only five with C (P=0 .01), In 10 patients who had previously received chemotherapy, none develop ed pattern C, five pattern B and five pattern A, in contrast to 15/41 patte rn C, 19/41 pattern B and 7/41 pattern A in healthy donors. These data sugg est an intrinsic loss of T-cell generation capacity from adult bone marrow stem cells in comparison to reports on stem cells of fetal origin. This los s correlated weakly with age, irrespective of thymic involution, and may be further reduced by prior chemotherapy.