Limiting-dilution analysis of long-term culture-initiating cells (LTCIC) is a quantitative method of estimating hematopoietic stem cell activity in clinical samples. We compared the numbers of LTCIC in bone marrow (BM), umbilical cord blood, and blood progenitor cells (obtained from patients with solid tumors at leukapheresis after mobilization with induction chemotherapy and filgrastim administration), using a two- stage long-term culture system and a limiting-dilution technique, scoring cobblestone areas of greater than 15 hematopoietic cells weekly for up to 8 weeks. Samples were obtained from 30 normal BMs, 20 human umbilical cords, and 32 leukapheresis products. Direct comparison of LTCIC in the three sources showed that the median proportions of cells generating hematopoietic foci from unfractionated mononuclear cells at 5 and 8 weeks, respectively, were 1:13,314 and 1:33,949 for BM, 1:12,506 and 1:34,546 for umbilical cord blood, and 1:10,302 and 1:12,891 for leukapheresis product. The estimated proportions of LTCIC from unfractionated mononuclear cells and CD34+ cells were similar in experiments with leukapheresis product. Leukapheresis product was superior to umbilical cord blood and cord blood to BM at 5 and 8 weeks of culture (P = .01). In two-stage long-term cultures, more colonies per flask and CD34+ cells were found in assays of leukapheresis product than in BM or umbilical cord blood cultures (P = .0005). Results obtained by this simplified limiting-dilution analysis correlated well with standard long-term cultures and can be used as a measure of the stem cell population. These data suggest that the incidence of putative stem cells in leukapheresis product and umbilical cord blood are at least comparable with that of BM.
Thirty-two patients with aplastic anemia (AA) have been studied using the long-term bone marrow culture (LTBMC) system. Of these patients, 26 had been treated with immunosuppressive therapy including antilymphocyte globulin (ALG) with or without androgens or high-dose methyl prednisolone. The remaining six patients either required no treatment or were studied before therapy was begun. Thirty-one of 32 patients (96%) had defective hematopoiesis in LTBMC with little or no evidence for the generation of primitive progenitor cells. The only exception was a patient with spontaneous recovery of aplasia in whom the defect was less marked. Crossover LTBMC experiments were performed in 23 cases by inoculating (1) patient marrow hematopoietic cells that had been depleted of adherent cells onto preformed, irradiated, normal stromas to assess the proliferative capacity of the hematopoietic cells, and (2) normal marrow hematopoietic cells that were depleted of adherent cells onto preformed, irradiated stromas from patients with AA to assess stromal function. Results of these experiments demonstrated a hematopoietic defect in all patients that was independent of the degree of hematologic recovery after ALG therapy. Only one patient had a probable stromal defect and this coexisted with a defect in the regenerative capacity of hematopoietic cells. We conclude that LTBMC is a sensitive method for detecting and defining the hematopoietic failure in AA. We suggest that the defective hematopoiesis present in all patients studied may be important in the pathogenesis of clonal evolution in AA.
Primitive hematopoietic cells released into the peripheral blood (PB) were studied in 50 patients with high-grade non-Hodgkin's lymphoma enrolled in a phase III trial of intensive weekly chemotherapy (VAPEC- B) alone or with granulocyte colony-stimulating factor (G-CSF). Mononuclear cells numbers were monitored and their in vitro growth potential assessed in clonogenic progenitor cell assays and in long- term culture. Total colony-forming cells (granulocyte-macrophage [GM], burst-forming unit, erythroid [BFU-E], Mix-CFC) were increased 40-fold (median) over baseline with chemotherapy alone and 106-fold with chemotherapy and G-CSF after the final dose. CD34+ cells were increased to a median of 4%, equivalent to that in normal bone marrow (BM) controls. Circulating colony-forming cell levels were maximal when the recovering total white blood cell (WBC) count reached 5 to 10 x 10(9)/L. The timing of the maximum was reproducible in individual patients. Therefore the WBC count can be used as a guide to the timing of leukapheresis. PB cells from normal controls' and patients' prechemotherapy were unable to sustain hemopoiesis in two-stage long- term cultures. In contrast, PB cells collected from patients primed with chemotherapy alone or chemotherapy with G-CSF at the time of predicted maximal colony-forming cell release were able to generate and sustain hematopoiesis in long-term cultures at a level comparable or superior to normal BM. These findings indicate that the use of G-CSF after routine outpatient chemotherapy stimulates maximal release of primitive hemopoietic cells into the circulation, including colony- forming cells and long-term culture-initiating cells. Their numbers are comparable with those in normal BM and are such that a single leukapheresis will usually yield enough cells for hemopoietic reconstitution after myeloablative chemotherapy.
Thirty-two patients with aplastic anemia (AA) have been studied using the long-term bone marrow culture (LTBMC) system. Of these patients, 26 had been treated with immunosuppressive therapy including antilymphocyte globulin (ALG) with or without androgens or high-dose methyl prednisolone. The remaining six patients either required no treatment or were studied before therapy was begun. Thirty-one of 32 patients (96%) had defective hematopoiesis in LTBMC with little or no evidence for the generation of primitive progenitor cells. The only exception was a patient with spontaneous recovery of aplasia in whom the defect was less marked. Crossover LTBMC experiments were performed in 23 cases by inoculating (1) patient marrow hematopoietic cells that had been depleted of adherent cells onto preformed, irradiated, normal stromas to assess the proliferative capacity of the hematopoietic cells, and (2) normal marrow hematopoietic cells that were depleted of adherent cells onto preformed, irradiated stromas from patients with AA to assess stromal function. Results of these experiments demonstrated a hematopoietic defect in all patients that was independent of the degree of hematologic recovery after ALG therapy. Only one patient had a probable stromal defect and this coexisted with a defect in the regenerative capacity of hematopoietic cells. We conclude that LTBMC is a sensitive method for detecting and defining the hematopoietic failure in AA. We suggest that the defective hematopoiesis present in all patients studied may be important in the pathogenesis of clonal evolution in AA.
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