This is probably because in both N/SD and N/N experimental combinations the nonadherent cells were derived from normal CD34 + cells (only the stromal layers were different), and since they were plated in equal numbers in the secondary short term cultures. Although CFU-GM colony numbers from N/SD nonadherent cells were lower than those from N/N nonadherent cells, the difference was not statistically significant. The production of CFU-GM colonies from the nonadherent cells in secondary clonogenic assays proved the existence of hematopoietic progenitors in the nonadherent cell population. The numbers of nonadherent cells harvested from N/SD LTCs from these cells in short-term cultures were clearly lower than the numbers obtained from the N/N LTCs. Differences between patients and controls were obvious. In our study, we assessed stromal support of hematopoiesis by first counting the nonadherent cells that were generated weekly, and also by looking at the clonogenic potential of these nonadherent cells in short-term clonogenic assays. This observation, which has not been previously reported, points to a more generalized marrow disorder than was formerly suspected. The second abnormal finding was a faulty marrow microenvironment. This dual defect exists in SD with and without myelodysplasia. We conclude that in addition to a stem-cell defect, patients with SD have also a serious, generalized marrow dysfunction with an abnormal bone marrow stroma in terms of its ability to support and maintain hematopoiesis. Stem-cell and stromal properties from the 2 patients with SD and myelodysplasia did not differ discernibly from SD patients without myelodysplasia. SD/N showed improved production of nonadherent cells and CFU-GM colonies compared with SD/SD, but much less than N/N. Nonadherent cells harvested weekly from N/SD LTCs were strikingly reduced compared with N/N LTCs numbers of granulocyte-monocyte colony-forming units (CFU-GM) derived from N/SD nonadherent cells were also lower. Normal marrow CD34 + cells were plated over either SD stroma (N/SD) or normal stroma (N/N) SD CD34 +cells were plated over either SD stroma (SD/SD) or normal stroma (SD/N). To assess marrow stromal function, long-term marrow stromal cell cultures (LTCs) were established. SD CD34 + cells plated directly in standard clonogenic assays showed markedly impaired colony production potential, underscoring an intrinsically aberrant progenitor population. Patients with SD had significantly lower numbers of CD34 + cells on bone marrow aspirates. To define the basis for the faulty hematopoietic function, 13 patients with SD (2 of whom had myelodysplasia with a clonal cytogenetic abnormality) and 11 healthy marrow donors were studied. Shwachman-Diamond syndrome (SD), an inherited disorder with varying cytopenias and a marked tendency for malignant myeloid transformation, is an important model for understanding genetic determinants in hematopoiesis.
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