In vitro proliferation and differentiation of megakaryocytic progenitors in patients with aplastic anemia, paroxysmal nocturnal hemoglobinuria, and the myelodysplastic syndromes

It has previously been shown that patients with aplastic anemia (AA) have a stem cell defect both of proliferation and differentiation. This has been shown by long-term bone marrow (BM) culture, long-term initiating cell assa ys, and committed progenitor assays. We present, for the first time, data o n megakaryocyte (Mk) colony formation from purified BM CD34(+) cells from p atients with AA. The results are compared with those from normal controls a nd from patients with paroxysmal nocturnal hemoglobinuria (PNH) and the mye lodysplastic syndromes (MDSs). Those treated for AA had previously received immunosuppression (antithymocyte globulin and/or cyclosporin). No patients had received bone marrow transplantation. A total of 13 AA patients (five untreated, eight treated), six PNH, six MDS, and 13 normal donors were stud ied. BM CD34(+) cells were purified by indirect labeling and then cultured in a collagen-based Mk assay kit (MegaCult-C, StemCell Technologies). The c ultures were fixed on day 12, and the Mk colonies were identified by the al kaline phosphatase anti-alkaline phosphatase technique using the monoclonal antibody CD41 (GP mdma). The slides were scored for Mk colony-forming unit s (CFU-Mks) (3-20 and >20 cells), Mk burst-forming units (BFU-Mks) (>50 cel ls), and mixed colonies. The results show that total Mk colony formation in AA was significantly low er than in normal donors (p < 0.0001), both in untreated patients/nonrespon ders to treatment (p = 0.0001) and in complete/partial responders (p < 0.00 2). There was no significant difference in Mk colony formation in treated a nd untreated patients (p = 0.05). Patients with AA had a lower total colony formation than PNH patients (p = 0.0002). PNH patients exhibited lower col ony formation than normal controls (p = 0.03), as shown by MDS patients, al though the considerable number of variables resulted in a lack of statistic ally significant difference from normal controls (p = 0.2), We have now shown that Mk colony formation from purified BM CD34(+) cells i s significantly reduced, supporting previous evidence that AA results from a stem cell defect.