Alterations in both the hematopoietic microenvironment and the progenitor cell population follow the recovery from myeloablative therapy and bone marrow transplantation

Abstract

Experimental data are conflicting, but suggest that after recovery from bone marrow transplantation (BMT), alterations in clonogenic growth and myeloid microenvironment remain. To further characterize this abnormality, light-density marrow cells from 15 patients who were in remission from hematologic malignancies and had undergone BMT (eight allogeneic and seven autologous) were studied in two culture systems after their marrows had reconstituted and were compared to normal. The preconditioning regimen for transplantation was fractionated total-body irradiation (TBI) 12 Gy, cyclophosphamide 120 mg/kg, and total nodal irradiation 6 Gy. Bone marrow mononuclear cells (MNCs) from the patients and controls were divided in two fractions; stromal layers (SL) were prepared from the first fraction. To examine the attributes of the stroma, the petri dish surface covered by the SL was measured after 5 weeks in culture, and representative layers were trypsinized and stained with Sudan black, butirate esterase, and acid and alkaline phosphatases. Stromal layers were also studied for their support in the development of blastic colonies (CFU-BI). From the second fraction, the CD34+ population was selected with immunomagnetic beads, and 1 x 10(4) progenitors from patients or controls were seeded onto the opposite group of preformed stroma. Stroma-adhesive precursors were scored for the formation of CFU-BI (> 20 cells) on day 5 of culture. Nonadherent selected CD34+ cells were recovered by standardized washing and quantitated in clonogenic assays (CFU-GM). The median patient age was 26 (SD 6.65) years, and eight of 15 patients were female. The median infused bone marrow (BM) MNC number was 0.9 x 10(8)/kg (SD 0.31). Grafts were studied at a median of 37 (SD 48.43) months from transplant. SL from the patients failed to reach confluence by 5 weeks (median dish area covered 55.5% [SD 32.38] vs. control: 100% [SD 2.35]; p = 0.0001). BMT CD34+ progenitors gave 19.5 (SD 42.2) CFU-Bl, significantly lower than those from normal individuals (127 [SD 62.2]; p = 0.01) when panned on control stroma, while control CD34+ cells were poorly supported on BMT layers (corrected for surface, median 2.5 [SD 42.2] CFU-Bl; p = 0.039). Although numbers of stroma nonadherent CFU-GM were not different between the groups (median BMT 56 [SD 54.5] vs. control 62.5 [SD 60.76]; p > 0.05), the ratio of CFU-Bl to CFU-GM showed a significant reduction in adherent CD34+ progenitors in BMT patients (median 0.28 [SD 0.44] vs. normal 2.09 [SD 1.3]; p = 0.04). None of the values were significantly different between patients receiving allogeneic or autologous grafts. We conclude that post-BMT stroma is defective and supports CFU-Bl growth poorly. Moreover, we documented a significant reduction within the CD34+ cells in the adherent primitive clonogenic precursors that was compensated by a proportional increase in the more mature CFU-GM.