Peripheral blood progenitor cell mobilisation alters myeloid, but not erythroid, progenitor cell self-renewal kinetics

Transplantation of progenitor cells which have been mobilised into the bloodstream (PBPC) following the administration of G-CSF results in more rapid neutrophil recovery than transplantation of bone marrow (BM). The reasons for the accelerated neutrophil engraftment are not clear, but would be explained by increased self-replication of myeloid progenitor cells (CFU-GM). We have used a CFU-GM replating assay to investigate myeloid progenitor self-replication, and quantification of subcolony formation during erythroid burst formation to quantify erythroid progenitor self-renewal. Secondary colony formation by CFU-GM, grown from PBPC and then replated was increased compared with secondary colony formation by BM CFU-GM (P = 0.0001); erythroid subcolony formation was not altered. There was no difference between the replating abilities of PBPC CFU-GM derived from allogeneic donors (normal individuals) and autologous donors (patients with malignant disease) although differences were found between subgroups of autologous donors. The increased replication of PBPC could not be accounted for by a reduction in progenitor cell apoptosis; PBPC CFU-GM contained slightly fewer apoptotic CD34+ cells than BM CFU-GM. The increased replication by PBPC CFU-GM was reversible because it declined when CFU-GM colonies were passaged through three sequential CFU-GM replating cycles. This decline in self-replication was more rapid than the decline seen in replated BM CFU-GM. The self-replication of PBPC CFU-GM, and subcolony formation by BFU-E could be further enhanced by exposure to cytokines in vitro. We conclude that mobilisation alters the replication kinetics of myeloid, but not of erythroid, progenitor cells, that mobilisation-induced events are of limited duration and that in vitro exposure to cytokines may modify PBPC progenitor cell kinetics.     

Evaluation of 'discordant maturation' in chronic myeloid leukaemia using cultures of primitive progenitor cells and their production of clonogenic progeny (CFU-GM)

The 'discordant maturation hypothesis' proposes that the most mature proliferating cells in chronic-phase chronic myeloid leukaemia (CML) are responsible for the expansion of the Ph-positive population. To evaluate this hypothesis we used a delta assay for primitive haemopoietic cells (PΔ assay for PΔ cells) which allows investigation of the kinetics of granulocyte-macrophage progenitor (CFU-GM) production. The frequencies of these primitive (PΔ) cells were similar in CML blood (14.5/10⁵ mononuclear cells), CML marrow (17.3/10⁵) and normal marrow (11.6/10⁵) The average frequency in normal blood is only 0.58/10⁶. The absolute numbers of PΔ cells in CML patients are therefore greatly increased. The average numbers of CFU-GM produced by individual PΔ cells were reduced in CML blood (8.1) and marrow (11.6) compared with normal marrow (28.5). This is consistent with a reduced probability of differentiation at the single cell level in CML. Although the absolute number of CFU-GM produced by individual CML PΔ cells was subnormal there was a relative increase in the number of day 7 CFU-GM compared with the number of day 14 and 21 CFU-GM, which agrees with the 'discordant maturation hypothesis'. This bias towards day 7 colony formation could reflect accelerated maturation by the CFU-GM produced by PΔ cells or, alternatively, the production of CFU-GM with shorter than normal maturation pathways. Overall, these results suggest that discordant maturation does not by itself account for myeloid expansion in CML. It is more likely that myeloid expansion in CML is due mainly to an increase in the number of primitive haemopoietic progenitor cells.     

Probability of progenitor renewal (PPR) and production of clonogenic progeny (CFU-GM) by primitive adherent progenitor cells in adult human bone marrow and umbilical cord blood

Summary. Human haemopoietic tissues contain primitive plastic-adherent progenitor cells (PΔ cells) that can be detected by measurement of their granulocyte-macrophage colony-forming cell (CFU-GM) progeny. Limiting dilution analysis and Poisson statistics are necessary for determining the frequency of PΔ cells because each of them produces several CFU-GM. Limiting dilution also permits measurement of the abilities of individual PΔ progenitors to produce CFU-GM. Here we report that the frequencies of PΔ progenitors in cord blood and adult marrow are similar (5.6 and 7.8/10⁵ mononuclear cells respectively) and individual cord blood PΔ progenitors produce fewer CFU-GM than adult PΔ progenitors. To test the possibility that the lower production of differentiated progeny by cord blood cells was the result of a higher rate of self-renewal, we devised a two-stage limiting dilution assay relying on the relative production of CFU-GM after two consecutive weeks of incubation. The probability of progenitor renewal (PPR) was derived from the number of wells (progenitors) that produced CFU-GM on both occasions compared with the number that produced CFU-GM on the first occasion only. The total number of CFU-GM produced on the second occasion compared with the number produced on the first occasion provided an index of the overall change in the size of the PΔ cell population. The data indicate that PΔ cells in cord blood have a higher PPR (0.59) than those in adult marrow (036). Also, the relative numbers of CFU-GM produced in the second and first weeks were greater for cord blood (1.2) than for adult marrow (0.36). Therefore PΔ cells in cord blood have a greater capacity for self-maintenance and possibly for expansion than PΔ cells in adult marrow.     

Feasibility of peripheral blood progenitor cell harvest and transplantation in patients with poor-risk myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are a group of clonal haematological disorders with a highly unfavourable prognosis. Allogeneic bone marrow transplantation offers the sole possibility for cure and prolonged survival, but is only available for a minority of patients. Therefore, we investigated the feasibility of PBPC collection and transplantation in 11 patients with high-risk myelodysplasia who were not eligible for allogeneic bone marrow transplantation. In six patients, PBPC were harvested after mobilization with G-CSF alone. Five patients were harvested during the recovery phase of intensive chemotherapy combined with G-CSF. This resulted in seven patients in an adequate CD34 progenitor yield >1 × 10⁶/kg. Six patients obtained a CFU-GM content of the PBPC harvest >10 ×10⁴/kg. Five patients were subsequently transplanted following a standard BuCy4 regimen. The median to ANC (absolute neutrophil count) ≥0.5 and 1.0 × 10⁹/l was respectively 14 d (range 10–18) and 16 d (range 11–25). Platelets were self-supporting ≥20 × 10⁹/l after a median of 41 d (range 8–144). One patient had a persistent lack of platelet engraftment unresponsive to infusion of back-up bone marrow.
These data demonstrate that in selected patients with high-risk MDS, adequate PBPC collection appears feasible, enabling the harvest of sufficient cell numbers required for rapid and stable engraftment after reinfusion. Improvement in mobilization efficiency may enable the collection of higher CD34⁺ progenitor cell numbers required for more rapid platelet engraftment. PBPC transplantation may be an alternative treatment option for patients who lack an allogeneic marrow donor. Follow-up is, however, still too limited to draw any conclusion regarding the long-term cure rate.     

Platelet-Derived Growth Factor Enhances Granulopoiesis Via Bone Marrow Stromal Cells

Platelet-derived growth factor (PDGF), a growth factor for connective tissue cells, stimulates erythropoiesis and megakaryocytopoiesis in vitro but the effect of PDGF on granulocyte proliferation remains unknown. The effect of the recombinant human PDGF-BB isoform on granulopoiesis was investigated in this study. The results show that PDGF significantly stimulated murine colony-forming unit-granulocyte-monocyte (CFU-GM) proliferation in a dose-dependent manner (1 to 100 ng/mL) using murine bone marrow cells (n = 4). Maximum stimulation was obtained with 50 ng/mL of PDGF (P < .01). The effect of PDGF on murine CFU-GM proliferation was compared with that of interleukin (IL)-3, IL-6, granulocyte-monocyte colony-stimulating factor (GM-CSF), and acidic fibroblast growth factor (aFGF) at their optimal doses. The stimulating activity of PDGF was higher than that of aFGF but lower than that of IL-3, IL-6, or GM-CSF. There is no synergistic effect between PDGF and IL-3 or IL-6, but a significant enhancing effect was observed in IL-3 plus IL-6. PDGF also stimulated the growth of CFU-GM with CFU-megakaryocyte in the presence of bone marrow stromal cells. We also found that PDGF had similar a effect on human CFU-GM proliferation using bone marrow mononuclear cells (MNC). However, the increase in PDGF-stimulated CFU-GM proliferation was inhibited by anti-GM-CSF, anti-IL-3, and anti-IL-6 antibodies (n = 4), suggesting that endogenously produced GM-CSF, IL-3, and IL-6 may play a role in the PDGF-induced CFU-GM proliferation. Furthermore, PDGF (1 to 100 ng/mL) did not show any effect on CFU-GM proliferation when replacing bone marrow MNC with immunomagnetic selection-enriched CD34+ cells from human cord blood (n = 5; purity, 91% +/- 6.5%). This study indicates that PDGF may indirectly enhance CFU-GM proliferation by inducing the bone marrow stromal cells to produce GM-CSF, IL-3, or IL-6.     

Purging of G-CSF-mobilized peripheral autografts in acute leukemia with mafosfamide and amifostine to protect normal progenitor cells

In the present study the in vitro growth of CFU-GM from PBPC of patients with AML (n = 11), purged with mafosfamide alone or a combination of mafosfamide and amifostine, was compared to historical controls of mafosfamide-purged bone marrow (AML CR1, n = 16). Two patients were transplanted with mafosfamide and mafosfamide/amifostine pretreated PBPC autografts. The in vitro experiments demonstrated a significantly higher resistance of peripheral blood derived CFU-GM to mafosfamide (median ID95 190 microg mafosfamide/ml) compared with bone marrow derived CFU-GM (median ID95130 microg/ml). Preincubation with amifostine significantly further increased the median ID95 to 245 microg/ml. The clinical results showed short recovery times for neutrophils >500/microl (9 and 13 days) and platelets >20 000/microl (12 and 21 days) and stable long-term engraftment with one relapse at day +118 and one patient in CR at day 760 after transplantation. The in vitro results show a significant advantage of PBPC over bone marrow-derived progenitors for purging with mafosfamide. Furthermore, a protective effect from mafosfamide of amifostine on normal progenitors could be demonstrated. The clinical results demonstrate the clinical feasibility of using mafosfamide-purged autologous PBPCT without impairing the short-term and long-term repopulating capacities of the autografts.     

Myeloablation with diaziquone: in vitro assessment

The promising antineoplastic agent diaziquone is associated with prolonged aplasia and rare instances of bone marrow necrosis, but only mild extramedullary toxicity. To explore the drug's potential as a myeloablative agent prior to bone marrow transplantation, we compared its effects on hematopoietic versus marrow stromal cells. After short- term (one to six hours) or prolonged (three to seven days) exposure to the drug, marrow was assayed for hematopoietic (CFU-Mix, BFU-E, CFU-GM) and stromal (CFU-F) colony-forming cells and studied in long-term marrow culture (LTMC). One- and three-hour treatments produced little cytotoxicity, even at 5000 ng/mL. After six-hour treatments with this dose, marrow was depleted of CFU-Mix, BFU-E, and CFU-GM, but produced CFU-GM in LTMCs, indicating an ongoing input of CFU-GM from a surviving pre-CFU-Mix population. In contrast, elimination of the latter may be inferred from the absence of CFU-GM in LTMCs exposed for three to seven days to diaziquone at only 150 ng/mL. Under these conditions, CFU-F recovery was 40% and adherent stromal layers in LTMCs were similar to untreated controls regarding rate of development and cellular composition. Our in vitro pre-CFU-Mix-ablative regimen correlates with clinical data that show prolonged but reversible myelosuppression at steady-state diaziquone plasma levels of 101 +/- 10 ng/mL (mean +/- standard error of mean) during 7-day constant infusions. In conclusion: hematopoietic cells are more sensitive than marrow stromal cells to the dose- and highly time-dependent cytotoxicity of diaziquone, a direct drug-induced noxious effect on the marrow microenvironment is an unlikely cause of the isolated episodes of marrow necrosis after the use of diaziquone in vivo, and prolonged infusion of diaziquone represents an attractive means for achieving myeloablation in selected clinical situations.     

Long-term haemopoiesis in human fetal liver cell cultures

Haemopoiesis in human fetal liver is almost entirely restricted to the erythroid series but when fetal liver cells were cultured under conditions established for the long-term maintenance of adult marrow haemopoiesis, a rapid switch to granulopoiesis was observed. Erythroid progenitor cells (BFU-E) rapidly disappeared, even though no humoral or cellular inhibitors of erythropoiesis could be detected, while myeloid progenitors (CFU-GM) increased in number. When the fetal liver cells were seeded onto stromal layers derived from adult marrow, in which endogenous haemopoiesis had ceased, granulopoiesis was established and maintained for more than a year, considerably longer than has previously been achieved with human haemopoietic cells.     

Hematopoietic recovery following high-dose combined alkylating-agent chemotherapy and autologous bone marrow support in patients in phase-I clinical trials of colony-stimulating factors: G-CSF,GM-CSF,IL-1, IL-2, M-CSF

Summary Hematopoietic recovery in 115 patients with metastatic breast cancer or metastatic melanoma, enrolled in phase-I studies of recombinant growth factors while undergoing treatment with high-dose chemotherapy with autologous bone marrow support, was examined with assays of bone marrow progenitor cells and peripheral blood progenitor cells, and by evaluation of peripheral blood counts. Groups of patients receiving hematopoietic cytokine support [with interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), or monocyte CSF (M-CSF)] post marrow infusion were compared with contemporaneous control patients not receiving growth factor support. Patients receiving GM-CSF demonstrated statistically significant increases in the growth of granulocyte/macrophage colony-forming units (CFU-GM) in the bone marrow and peripheral blood compared with control patients. The effect of GM-CSF was dose dependent in the early period post marrow infusion (day +6) with bone marrow CFU-GM colonies at doses 8–16 g/kg/ day 34 times those measured in controls. Significant increases in bone marrow multipotential progenitor cells (CFU-GEMM) were seen in patients receiving GMCSF day + 21 post marrow infusion. Patients receiving IL-1 demonstrated significant increases in bone marrow CFU-GM at day +21, maximal at dosages of 24–32 ng/kg/day. There were no significant increases in burst forming unit-erythroid (BFU-E) among any study group. Patients receiving G-CSF had significantly increased absolute neutrophil counts (ANC) and total white blood cell counts (WBC) by day +11 post transplant compared with control patients. Patients receiving GM-CSF demonstrated significantly increased WBC (greater than 2000/mm³) at day +11 and ANC greater than 500/mm³ at day +16. Optimal dose of GCSF and GM-CSF to stimulate neutrophil recovery post transplant was 4–8 g/kg/day and 8–16 g/kg/day, respectively. Platelet recovery did not differ among the six study groups. These data demonstrate accelerated myeloid recovery after high-dose chemotherapy and autologous bone marrow support in patients receiving either G-CSF or GM-CSF. Moreover, GM-CSF and IL-1 stimulate myelopoiesis at the level of bone marrow CFU-GM, while G-CSF causes earlier neutrophil recovery peripherally.This work has been supported in part by The National Heart, Lung, and Blood Institute, grant P01CA47741. Joanne Kurtzberg, MD is a scholar of the Leukemia Society of America     

Normal and malignant human myeloid progenitors differ in their sensitivity to hyperthermia

The effect of in vitro hyperthermia on normal human bone marrow granulocyte-macrophage progenitor cells (granulocyte-macrophage colony-forming units, CFU-GM) was compared to its effect on clonogenic acute nonlymphocytic leukemic (ANLL) cells. Mononuclear normal bone marrow cells, blasts from patients with ANLL, and HL-60 cells were incubated at room temperature (control) and at 42 degrees-44 degrees C for 0-120 min prior to assay in methylcellulose. The heat sensitivity of the leukemic cells was significantly greater than that of normal bone marrow progenitors. Two-h exposure to 43 degrees C, for example, resulted in survival of 52% of normal marrow CFU-GM, whereas only 3% of leukemic CFU-GM survived (p less than 0.001 for HL-60 cells and p less than 0.005 for patient blast cells). To determine the effect of hyperthermia on more primitive progenitors and on marrow stromal cells, long-term cultures of normal bone marrow were established using control and heat-treated cells. Generation of CFU-GM was detected in the nonadherent fraction of hyperthermia-treated samples throughout the 5-week culture period. Although stromal development was slightly delayed, hyperthermia-treated cells were able to establish stromal layers similar to control cells. These results indicate that normal bone marrow committed progenitor cells are more resistant to hyperthermia than are myeloid leukemic cells. Normal stromal cells and primitive cells assayed in long-term culture are also resistant to hyperthermia that is toxic for leukemic cells. Because of this differential sensitivity to heat, ex vivo hyperthermia may be applicable for removing residual leukemic cells from bone marrow harvested for autologous transplantation.     

Progenitor cell assays predict hematopoietic reconstitution after syngeneic transplantation in mice

Hematopoietic reconstitution following syngeneic bone marrow transplantation with graded doses of untreated and drug-treated bone marrow was studied in B6D2F1 mice. Granulocyte-macrophage colony- forming units (CFU-GM) and spleen colony-forming units (CFU-S) showed similar in vitro drug sensitivities. Both the speed of hematologic recovery and survival of mice transplanted with untreated or drug- treated bone marrow were directly related to the number of CFU-GM or CFU-S transplanted. Similar hematologic recovery was seen for untreated marrow transplants and treated transplants that had similar CFU-GM or CFU-S content. There is a minimum number of transplanted CFU-GM or CFU- S that allows survival of lethally irradiated mice. This number is present in a marrow transplant containing the equivalent of 5 X 10(3) untreated cells or producing one to two spleen colonies. There also exists a maximum value for the number of hematopoietic progenitors in a marrow graft, above which the rate of hematologic recovery following transplantation is rapid and no detectable increase in the rate is seen with increasing CFU-GM or CFU-S content. The presence of this maximum value for transplanted progenitors and variations in culture techniques are probably the reasons previous studies have not always shown a correlation between CFU-GM content and hematologic recovery after bone marrow transplantation.     

Long-lasting decrease of marrow and circulating long-term culture initiating cells after allogeneic bone marrow transplant.

We investigated bone marrow (BM) and circulating (PB) hematopoietic progenitor cells in 37 normal donors and in 25 patients 1 to 8 years after successful allogeneic bone marrow transplant. At the time of testing, transplanted patients had normal blood counts and bone marrow cellularity. By flow cytometry, BM CD34+ cells were found to be three- to four-fold decreased in transplanted patients compared to normal donors, while the number of PB CD34+ cells was the same as in normal donors. Using a methylcellulose colony assay, primary BM colony-forming cells (CFU-GM) were decreased 2.1-fold, whereas PB CFU-GM were only marginally decreased. In a long-term culture initiating cell (LTC-IC) assay, an eight-fold decrease of early progenitor cells was observed in the marrow of transplanted patients compared to normal donors, and a five-fold decrease was documented in peripheral blood. We found that the BM LTC-IC cell number correlated with concurrently determined BM CD34+ cells and committed progenitor cell number (measured as CFU-GM) and with PB LTC-IC number, but not with PB CFU-GM and CD34+ cells. We conclude that marrow and circulating early stem cell compartments, as measured by the LTC-IC assay, are greatly and permanently depressed following bone marrow transplant. The correlation between BM and PB LTC-IC indicates that the enumeration of circulating LTC-IC can be used as a measure of the stem cell compartment in the bone marrow after transplant. It seems that the deficiency of the most immature progenitor cells persists forever after successful bone marrow transplant; this means that a complete hematopoietic reconstitution can be sustained by a reduced stem cell pool.     

Effect in vivo of recombinant GM-CSF on neutropenia and survival in mice treated by high-dose melphalan

High doses of melphalan cause severe neutropenia and may irreversibly damage hematopoietic stem cells. Treatment of mice with recombinant murine GM-CSF (GM-CSF) for 5 days immediately after 400 micrograms of melphalan did not prevent the severe neutropenia. However, GM-CSF accelerated the neutrophil recovery and reduced the mortality rate during the neutropenic period compared to melphalan-only treated mice. CFU-GM levels measured 6 d after melphalan treatment without GM-CSF were markedly reduced in the bone marrow while being elevated in the spleen. In comparison, GM-CSF further reduced the total CFU-GM population in melphalan-treated mice including the levels in the bone marrow and in the spleen. On d 14 after melphalan, the spleen regained its active CFU-GM production. By d 90, the number of circulating neutrophils, the number of bone marrow CFU-GM and splenic CFU-GM were the same in GM-CSF-treated and -untreated mice. The results suggest that GM-CSF could be used to shorten the neutropenic period and reduce mortality caused by a high dose of melphalan. Though this effect could be at the expense of a temporary reduction in CFU-GM population, GM-CSF did not induce more long-term damage to myelopoiesis than that already caused by melphalan alone.     

Ex-vivo expansion of bone marrow progenitor cells for hematopoietic reconstitution following high-dose chemotherapy for breast cancer

The use of hematopoietic growth factors, stromal monolayers, and frequent medium exchange allows the expansion of hematopoietic progenitors ex-vivo. We evaluated the use of ex-vivo expanded progenitor cells for hematopoietic reconstitution following high dose chemotherapy (HDC) in breast cancer patients. Patients with high-risk Stage II or metastatic breast carcinoma underwent bone marrow aspirations using general anesthesia. A total of 675-1125 x 10(6) mononuclear cells (MNC) were seeded for ex-vivo expansion for 12 days in controlled perfusion bioreactors (Aastrom Biosciences, Inc.). The bone marrow cultures, which included the stromal cells collected with the aspirate, were supplemented with erythropoietin, granulocyte-macrophage-colony stimulating factor (GM-CSF)/IL-3 fusion protein (PIXY 321), and flt3 ligand. Stem cell transplant was performed with expanded cells after HDC. A median bone marrow volume of 52.9 mL (range 42-187 mL) was needed to inoculate the bioreactors. Median fold expansion of nucleated cells (NC) and colony forming unit granulocyte-macrophage (CFU-GM) was 4.9 and 9.5, respectively. The median fold expansion of CD34+lin- and long-term culture-initiating culture (LTC-IC) was 0.42 and 0.32, respectively. Five patients were transplanted with ex-vivo expanded NC. Median days to an absolute neutrophil count > 500/microL was 18 (range 15-22). Median days to a platelet count > 20,000/microl was 23 (range 19-39). All patients had sustained engraftment of both neutrophils and platelets. Immune reconstitution was similar to that seen after HDC and conventional stem cell transplantation. We conclude that ex-vivo expansion of progenitor cells from perfusion cultures of small volume bone marrow aspirates, allows hematopoietic reconstitution after HDC.     

Comparison of megakaryopoiesis in vitro of paired peripheral blood progenitor cells and bone marrow harvested during remission in patients with acute myeloid leukaemia

We have studied paired peripheral blood progenitor cells (PBPC) and bone marrow (BM) samples from 12 acute myeloid leukaemia (AML) patients following intensive chemotherapy, and assessed direct granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), megakaryocyte CFU (CFU-Mk) numbers and the production of CD61+ (platelet glycoprotein IIIa) cells in suspension culture in response to various haemopoietic growth factor combinations. We found that CFU-GM and BFU-E numbers per 105 mononuclear cells were similar in both AML PBPC and BM harvests; CFU-Mk numbers, however, were significantly higher in PBPC than BM. In addition, the higher total white cell count of the PBPC harvests meant that PBPC have much higher numbers of total progenitors per collection. CD61+ cell numbers in suspension cultures of AML PBPC and BM were lower than those of harvested normal marrow. However, response to pegylated recombinant human megakaryocyte growth and development factor (PEGrHuMGDF) both alone and in combination with other growth factors was qualitatively similar to that of normal BM. As with normal BM, response to PEGrHuMGDF alone did not increase further with addition of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), interleukin 6 (IL-6) or erythropoietin (EPO) in the AML PBPC and BM. Further responses over PEGrHuMGDF alone were seen when added with stem cell factor (SCF) or with a combination of SCF + IL-3 + EPO in both AML PBPC and BM cultures; however, the magnitude of the response was greater in the PBPC cultures. Response to PEGrHuMGDF + IL-3 was seen in the PBPC cultures but not in the AML BM. These data suggest that, in AML patients, there are proportionally more megakaryocyte progenitor cells in the mobilized PBPC than in the BM harvests, which would explain the more rapid platelet recovery following PBPC autografts.     

Autologous progenitor cell transplantation: prior exposure to stem cell- toxic drugs determines yield and engraftment of peripheral blood progenitor cell but not of bone marrow grafts

Agents with stem cell-toxic potential are frequently used for salvage therapy of Hodgkin's disease (HD) and high-grade non-Hodgkin's lymphoma (NHL). Because many patients with relapsed or refractory lymphoma are candidates for autologous progenitor cell transplantation, possible toxic effects of salvage chemotherapy on progenitor cells must be taken into account. In a retrospective study, we have analyzed the influence of a salvage regimen containing the stem cell-toxic drugs BCNU and melphalan (Dexa-BEAM) on subsequently harvested bone marrow (BM)- and peripheral blood-derived progenitor cell grafts (PBPC) and compared it with other factors. Progenitor cells were collected from 96 patients with HD or high-grade NHL. Seventy-nine grafts were reinfused (35 PBPC and 44 BM) after high-dose chemotherapy. Compared with patients autografted with BM, hematopoietic recovery was significantly accelerated in recipients of PBPC. For PBPC, the number of Dexa-BEAM cycles ( > or = v > 1) was the predominate prognostic factor affecting colony-forming unit-granulocyte-macrophage (CFU-GM) yield (66 v 6.8 x 10(4)/kg, P = .0001), CD34+ cell yield (6.6 v 1.6 x 10(6)/kg, P = .0001), neutrophil recovery to > 0.5 x 10(9)/L (9 v. 11 days, P = .0086), platelet recovery to > 20 x 10(9)/L (10 v 15.5 days, P = .0002), and platelet count on day +100 after transplantation (190 v 107 x 10(9)/L, P = .031) using univariate analysis. Previous radiotherapy was associated with significantly lower CFU-GM and CD34+ cell yields but had no influence on engraftment. Patient age, patient sex, disease activity, or chemotherapy other than Dexa-BEAM did not have any prognostic impact. Multivariate analysis confirmed that Dexa-BEAM chemotherapy was the overriding factor adversely influencing CFU-GM yield (P < .0001), CD34+ cell yield (P < .0001), and platelet engraftment (P < .0001). BM grafts were not significantly affected by previous Dexa-BEAM chemotherapy or any other variable tested. However, prognostic factors favoring the use of BM instead of PBPC were not identified using joint regression models involving interaction terms between the graft type (PBPC or BM) and the explanatory variables investigated. We conclude that, in contrast to previous radiotherapy or other chemotherapy, exposure to salvage regimens containing stem cell- toxic drugs, such as BCNU and melphalan, is a critical factor adversely affecting yields and performance of PBPC grafts. Marrow progenitor cells appear to be less sensitive to stem cell-toxic chemotherapy. PBPC should be harvested before repeated courses of salvage chemotherapy involving stem cell-toxic drugs to preserve the favorable repopulation kinetics of PBPC in comparison with BM.     

Long-term human bone marrow cultures

Pronlonged in vitro growth of murine bone marrow has been achieved using a flask culture system. We report the adaption of the technique to the growth of human bone marrow. In this system, CFU-GM and CFU-E are maintained for 4-9 wk. Morphologically recognizable granulopoiesis occurs for 4-6 wk and erythropoiesis for 1.5-2.5 wk. Functional T lymphocytes are maintained for at least 5 wk. The adherent population is composed of macrophages, fibroblast-like cells, and flat pavement- like cells. Fat-containing cells are not prominant. This culture system provides a means to study human hematopoietic cell proliferation and differentiation in vitro, as well as to examine interactions between stromal cells and hematopoietic and lymphoid progenitors.     

Immune reconstitution following transplantation of autologous peripheral CD34+ cells.

The recovery of lymphocyte count, CD4+ and CD8+ T-cell subsets, natural killer (NK) cells and CD19+ B cells has been evaluated during the first 4 months after the infusion of autologous CD34+ peripheral blood progenitor cells (PBPC; group A; 33 patients) or autologous unselected PBPC (group B; 36 patients) for hematological malignancies. Lymphocyte count promptly recovered in both patient cohorts, although the repopulation of CD3+ T cells occurred more rapidly in group B compared with group A. The count of CD4+ T lymphocytes remained <200/microl during the study period in patients transplanted with CD34+ PBPC, being significantly lower compared with group B (p = 0.0019 and p = 0.0035 on days 30 and 60, respectively). CD8+ T cells rapidly increased both in group A and B and CD4 to CD8 ratio was severely reduced. CD4+ and CD8+ T cells displayed an activated phenotype in both groups of patients, coexpressing the HLA-DR antigen throughout the study period. No differences in the repopulation kinetics of NK cells and CD19+ B cells were observed. Further investigations are encouraged to characterize T cell competence following transplantation of CD34+ PBPC.     

Sustained long-term hematopoiesis after myeloablative therapy with peripheral blood progenitor cell support

A retrospective analysis of long-term hematopoiesis was performed in a group of 145 consecutive patients who had received high-dose therapy with peripheral blood progenitor cell (PBPC) support between May 1985 and December 1993. Twenty-two patients had acute myelogenous leukemia, nine had acute lymphoblastic leukemia, 43 had Hodgkin's disease, 57 had non-Hodgkin's lymphoma, and 14 patients had multiple myeloma. Eighty- four patients were male and 61 female, with a median age of 37 years (range, 16 to 58 years). In 46 patients, PBPC were collected after cytotoxic chemotherapy alone, while 99 patients received cytokines either during steady-state hematopoiesis or post-chemotherapy. Sixty patients were treated with dose-escalated polychemotherapy, and 85 patients had a conditioning therapy including hyperfractionated total body irradiation at a total dose of 14.4 Gy. The duration of severe pancytopenia posttransplantation was inversely related to the number of reinfused granulocyte-macrophage colony-forming units (CFU-GM) and CD34+ cells. Threshold quantities of 2.5 x 10(6) CD34+ cells per kilogram or 12.0 x 10(4) CFU-GM per kilogram became evident and were associated with rapid neutrophil and platelet recovery within less than 18 and 14 days, respectively. These numbers were also predictive for long-term reconstitution, indicating that normal blood counts are likely to be achieved within less than 10 months after transplantation. Conversely, 12 patients were autografted with a median of 1.75 x 10(4) CFU-GM per kilogram resulting in delayed recovery to platelet counts of greater than 150 x 10(9)/L between 1 and 6 years. Our study includes bone marrow examinations in 50 patients performed at a median follow-up time of 10 months (range, 1 to 85 months) posttransplantation. A comparison with normal volunteers showed a 3.2-fold smaller proportion of bone marrow CD34+ cells, which was paralleled by an even more pronounced reduction in the plating efficiency of CFU-GM and burst- forming unit-erythroid. No secondary graft failure was observed, even in patients autografted with relatively low numbers of progenitor cells. This suggests that either the pretransplant regimens were not myeloablative, allowing autochthonous recovery, or that a small number of cells capable of perpetual self-renewal were included in the autograft products.     

Granulopoietic differentiation in long-term bone marrow cultures from children with congenital neutropenia

The capacity of granulopoietic precursor cells (CFU-GM) to differentiate in vitro was evaluated in five children with congenital neutropenia using short-term colony assays and long-term marrow cultures. In all five children, methylcellulose assays revealed normal numbers of CFU-GM, which displayed an appropriate response to various sources of GM-CSF and differentiated up to the polymorphonuclear leukocyte state (PMN). In contrast, neutrophil PMN were not observed in long-term bone marrow cultures from three patients, despite a normal production of CFU-GM, myeloblasts, and promyelocytes during the 5-6 week culture period. Thus, in these patients, the characteristic "block" in granulocytic maturation observed in vivo was reproduced in vitro in long-term cultures. Granulocytic differentiation proceeded normally in long-term cultures from the two other patients, thus indicating heterogeneity in the expression of the defect. These results might indicate abnormal interactions between stromal and hematopoietic cells in long-term marrow cultures from some patients with congenital neutropenia. Furthermore, our results showed some correlation between the granulocytic defect in vitro and the clinical outcome in vivo.