A comparative study of the phenotype and proliferative capacity of peripheral blood (PB) CD34+ cells mobilized by four different protocols and those of steady-phase PB and bone marrow CD34+ cells

Peripheral blood (PB) CD34+ cells from four commonly used mobilization protocols were studied to compare their phenotype and proliferative capacity with steady-state PB or bone marrow (BM) CD34+ cells. Mobilized PB CD34+ cells were collected during hematopoietic recovery after myelosuppressive chemotherapy with or without granulocyte- macrophage colony-stimulating factor (GM-CSF) or granulocyte colony- stimulating factor (G-CSF) or during G-CSF administration alone. The expression of activation and lineage-associated markers and c-kit gene product were studied by flow cytometry. Proliferative capacity was measured by generation of nascent myeloid progenitor cells (granulocyte- macrophage colony-stimulating factor; CFU-GM) and nucleated cells in a stroma-free liquid culture stimulated by a combination of six hematopoietic growth factors (interleukin-1 (IL-1), IL-3, IL-6, GM-CSF, G-CSF, and stem cell factor). G-CSF-mobilized CD34+ cells have the highest percentage of CD38- cells (P < .0081), but otherwise, CD34+ cells from different mobilization protocols were similar to one another in their phenotype and proliferative capacity. The spectrum of primitive and mature myeloid progenitors in mobilized PB CD34+ cells was similar to their steady-state counterparts, but the percentages of CD34+ cells expressing CD10 or CD19 were lower (P < .0028). Although steady-state PB and chemotherapy-mobilized CD34+ cells generated fewer CFU-GM at day 21 than G-CSF-mobilized and steady-state BM CD34+ cells (P < .0449), the generation of nucleated cells and CFU-GM were otherwise comparable. The presence of increased or comparable numbers of hematopoietic progenitors within PB collections with equivalent proliferative capacity to BM CD34+ cells is not unexpected given the rapid and complete hematopoietic reconstitution observed with mobilized PB. However, all four types of mobilized PB CD34+ cells are different from steady-state BM CD34+ cells in that they express less c-kit (P < .0002) and CD71 (P < .04) and retain less rhodamine 123 (P < .0001). These observations are novel and suggest that different mobilization protocols may act via similar pathways involving the down-regulation of c-kit and may be independent of cell-cycle status.     

In vitro effect of stem cell factor on human clonogenic marrow progenitors after myeloablative treatments

Abstract: Abnormal hematopoiesis, including a deficiency of marrow progenitors and particularly of erythroid progenitors, has been described after autologous stem cell transplantation (ASCT), persisting for several years. In order to explain this deficiency, a resistance of marrow progenitors to stem cell factor (SCF) after ASCT was investigated. Marrow samples were harvested from pregraft patients at graft collection prior to ASCT, transplanted patients 6–24 months after high-dose therapy and control patients. CD34+ cells were cultured in a serum-free clonogenic assay with increasing doses of SCF. The clonogenic efficiency without SCF was lower for BFU-E in treated groups than in controls, whereas it was not different for CFU-GM. With increasing doses of SCF a dose-dependent effect was found on the numbers of both CFU–GM and BFU–E in all groups, although the maximal number of BFU–E remained lower in treated groups. However, the SCF dose that induced 50% of maximal BFU–E growth (D₅₀) was similar in all groups. Furthermore, a dose-dependent effect on the size of BFU–E was found in all groups, with no difference in the proportion of large colonies. Thus, clonogenic erythroid progenitors from patients who have received myelotoxic treatments remain sensitive to SCF, with no evidence for a chemotherapy-related resistance.     

Haematopoietic action of flt3 ligand on cord blood-derived CD34-positive cells expressing different levels of flt3 or c-kit tyrosine kinase receptor: comparison with stem cell factor

We compared the effect of human flt3 ligand (FL) and stem cell factor (SCF) on cord blood (CB)-derived CD34+ cells expressing different levels of flt3 or c-kit tyrosine kinase (TK) receptor in clonal cell culture. The c-kit receptor was expressed by 58.5±16.7% of CB CD34+ cells (n = 19), in which c-kit^high, c-kit^low and c-kit^- cell populations could be identified. In contrast, the flt3 receptor (FR) was weakly expressed on 58.6±8.3% (n = 9) of CB CD34+ cells. FL+erythropoietin (Epo) failed to support erythroid burst (BFU–E) formation by any subpopulation of CD34+ cells. However, SCF+Epo supported BFU–E and erythrocyte-containing mixed (CFU–mix) colony formation from all subpopulations. Interestingly, FL markedly augmented CFU–mix colony formation supported by interleukin (IL)- 3+Epo when CD34+c-kit^low or CD34+FR+ cells were used as the target. On the other hand, SCF significantly enhanced CFU-mix colony formation supported by IL-3+Epo when CD34+c-kit^{high or low} and CD34+FR+ cells were used. The replating potential of CFU–mix supported by IL-3 + Epo + FL was greater when CD34+c-kit^low or CD34+FR+ cells were used. When the CD34+c-kit^low cells were used, the number of lineages expressed in secondary cultures of CFU–mix colonies derived from primary cultures containing IL-3 + Epo+FL or SCF was significantly larger than when the primary cultures contained IL-3+Epo. Furthermore, the number of long-term culture-initiating cells found in CD34+FR+ cells was larger than that in FR^- cells. CB-derived CD34+c-kit^low cells represent a less mature population than c-kit^high cells, as reported previously. Therefore, these results indicate that both FL and SCF can act on primitive multipotential progenitors. However, it is still uncertain whether CB-derived CD34+FR+ cells are less mature than CD34+FR^- cells.     

Human FLT3 ligand acts on myeloid as well as multipotential progenitors derived from purified CD34+ blood progenitors expressing different levels of c-kit protein

Abstract: We studied the effect of human flt3/flk2 ligand (FL) on the proliferation and differentiation of purified CD34⁺ blood progenitors which express different levels of c-kit protein in clonal cell culture in comparison with that of stem cell factor (SCF). FL alone did not support significant colony formation. However, FL significantly enhanced neutrophil colony (CFU–G) formation in the presence of granulocyte-colony stimulating factor (G–CSF) by peripheral blood (PB)-derived CD34⁺c-kit^? cells which contained a large number of CFU–G. In addition, FL could synergistically increase the number of CFU–G supported by a combination of interleukin (IL)-3 and G–CSF, as did SCF. As we reported previously, SCF showed a significant burst-promoting activity (BPA). In contrast, FL did not exhibit any BPA on PB-derived CD34⁺c-kit^high cells in which erythroid-burst (BFU-E) was highly enriched. However, FL could synergize with IL-3 or GM–CSF in support of erythrocyte-containing mixed (E-Mix) colony by PB-derived CD34⁺c-kit^{high or low} cells in the presence of Epo. Replating of E-Mix colonies derived from CD34⁺c-kit^high cells supported by IL-3+Epo+SCF yielded more secondary colonies than those supported by IL-3+Epo or IL-3+Epo+FL. When PB-derived CD34⁺c-kit^low cells which represent a more immature population than CD34⁺c-kit^high cells were used as the target, number of secondary colonies supported by IL-3+Epo, IL-3+Epo+SCF or IL-3+Epo+FL was comparable. However, the number of lineages expressed in the secondary culture was significantly larger in the primary culture containing IL-3+Epo+FL than in that containing IL-3+Epo. These results suggest that FL not only acts on neutrophilic progenitors, but also on more immature multipotential progenitors.     

Peripheral blood stem cell (PBSC) mobilization and transplantation after fludarabine therapy in chronic lymphocytic leukaemia (CLL): a report of the European Blood and Marrow Transplantation (EBMT) CLL subcommittee on behalf of the EBMT Chronic Leukaemias Working Party (CLWP)

We performed a survey from 122 centres of the European Group of Blood and Marrow Transplantation (EBMT) concerning peripheral blood stem cell (PBSC) mobilization after fludarabine treatment of patients with chronic lymphocytic leukaemia (CLL). A total of 101 leucaphereses from 29 patients was performed. The median cell numbers collected were: CD34+ cells, 2.2 x 106/kg (0.1-15.3); granulocyte-macrophage colony-forming units (GM-CFU), 4.29 x 104/kg (0.4-177); and mononuclear cells, 6.4 x 108/kg (1.3-63). In univariate and multivariate analyses, the numbers of cells collected were not significantly influenced by the nature of mobilizing regimen and there was a trend towards the collection of a higher number of CD34+ cells from patients who received fludarabine only before mobilization. There was a significant correlation between the median number of CD34+ cells collected and the number of courses of fludarabine (higher CD34+ cell numbers were related to more than six courses) and the interval between the last dose of fludarabine and the start of mobilizing therapy (higher CD34+ cell numbers were related to a delay > or = 2 months). Sixteen patients have subsequently undergone autologous transplantation and showed rapid engraftment. In conclusion, the results reported favour early stem cell mobilization in CLL patients who are in remission after first-line therapy. However, attention should be given to the timing of mobilization with respect to the time since the last dose of fludarabine.     

Geldanamycin and herbimycin A induce apoptotic killing of B chronic lymphocytic leukemia cells and augment the cells' sensitivity to cytotoxic drugs

We studied the actions of geldanamycin (GA) and herbimycin A (HMA), inhibitors of the chaperone proteins Hsp90 and GRP94, on B chronic lymphocytic leukemia (CLL) cells in vitro. Both drugs induced apoptosis of the majority of CLL isolates studied. Whereas exposure to 4-hour pulses of 30 to 100 nM GA killed normal B lymphocytes and CLL cells with similar dose responses, T lymphocytes from healthy donors as well as those present in the CLL isolates were relatively resistant. GA, but not HMA, showed a modest cytoprotective effect toward CD34+ hematopoietic progenitors from normal bone marrow. The ability of bone marrow progenitors to form hematopoietic colonies was unaffected by pulse exposures to GA. Both GA and HMA synergized with chlorambucil and fludarabine in killing a subset of CLL isolates. GA- and HMA-induced apoptosis was preceded by the up-regulation of the stress-responsive chaperones Hsp70 and BiP. Both ansamycins also resulted in down-regulation of Akt protein kinase, a modulator of cell survival. The relative resistance of T lymphocytes and of CD34+ bone marrow progenitors to GA coupled with its ability to induce apoptosis following brief exposures and to synergize with cytotoxic drugs warrant further investigation of ansamycins as potential therapeutic agents in CLL.     

Colony formation of clone-sorted human hematopoietic progenitors.

To characterize human hematopoietic progenitors, we performed methylcellulose cultures of single cells isolated from a population of CD34+ cells by fluorescence-activated cell-sorting (FACS) clone-sorting system. CD34+ cells were detected in bone marrow (BM) and peripheral blood (PB) cells at incidences of 1.0% and 0.01% of total mononuclear cells, respectively. Single cell cultures revealed that approximately 37% of BM CD34+ cells formed colonies in the presence of phytohemagglutinin-leukocyte conditioned medium and erythropoietin. Erythroid bursts-, granulocyte-macrophage (GM) colony-, and pure macrophage (Mac) colony-forming cells were 10% each in CD34+ cells. Approximately 15% of PB CD34+ cells formed colonies in which erythroid bursts were predominant. CD34+ cells were heterogeneous and fractionated by several antibodies in FACS multicolor analysis. In these fractionated cells, CD34+, CD33+ cells formed GM and Mac colonies 7 to 10 times as often as CD34+, CD33- cells. Most of the erythroid bursts and colonies were observed in the fraction of CD34+, CD13- cells or CD34+, CD33- cells. The expression of HLA-DR on CD34+ cells was not related to the incidence, size, or type of colonies. There was no difference in the phenotypical heterogeneity of CD34+ cells between BM and PB. About 10% of CD34+ cells were able to form G colonies in response to granulocyte colony-stimulating factor (G-CSF) and to form Mac colonies in GM-CSF or interleukin-3 (IL-3). Progenitors capable of generating colonies by stimulation of G-CSF were more enriched in CD34+, CD33+ fraction than in CD34+, CD33- fraction. Thus, single cell cultures using the FACS clone-sorting system provide an accurate estimation of hematopoietic progenitors and an assay system for direct action of colony-stimulating factors.     

Possibility of progenitor cell mobilization during the hematological recovery following peripheral blood stem cell autograft

Twenty-four patients with hematological malignancies were studied during recovery following autografting in order to establish the proportion of patients that show CD34+ cell mobilization and the kinetics of mobilized CD34-positive cells. The patients showed a peak in peripheral blood (PB) CD34+ cells after a median of 14 days (range 12-20) following reinfusion. According to the number of circulating CD34+ cells, two groups could be clearly distinguished: 17 patients (group A) with <10 PB CD34+ cells/microl (median 1.2, range 0-5) and 7 patients (group B) with >10 CD34+ cells/microl (median 51, range 13-123). Compared to group A, patients of group B showed a faster hematological reconstitution of both polymorphonuclear leukocytes >500/microl (12 vs. 15 days) and platelets >50,000/microl (12 vs. 17 days). The expression of the beta1 integrin CD49d was similar in the two groups of patients, while a lower expression of the beta2 integrin CD11a and a greater expression of the L-selectin CD62L were observed in the PB CD34+ cells of group B patients. Both in the PB and in the BM, the number of CFU-GEMM, CFU- GM, CFU-E and BFU-E of group B was significantly greater than that of group A. However, when the clonogenic potential of a single CD34+ cell was evaluated, no major differences in the number of colonies produced per CD34+ cell were found between the two groups.     

Characterization of CD34+ subsets derived from bone marrow, umbilical cord blood and mobilized peripheral blood after stem cell factor and interleukin 3 stimulation

We characterized CD34+ cells purified from bone marrow (BM), mobilized peripheral blood (PB) and cord blood (CB) and we tried to establish correlations between the cell cycle kinetics of the CD34+CD38- and CD34+CD38+ subpopulations, their sensitivity to SCF and IL-3 and their expression of receptors for these two CSFs. At day 0, significantly fewer immature CD34+CD38- cells from CB and mobilized PB are in S + G2M phases of the cell cycle (respectively 2.0 +/- 0.4 and 0.9 +/- 0.3%) than their BM counterpart (5.6 +/- 1.2%). A 48-h incubation with SCF + IL-3 allows a significant increase in the percentage of cycling CD34+CD38- cells in CB (19.2 +/- 2.2%, P < 0.0002) and PB (14.1 +/- 5.5%, P < 0.05) while the proliferative potential of BM CD34+CD38- progenitors remains constant (8.6 +/- 1.0%, NS). CD123 (IL-3 receptor) expression is similar in the three sources of hematopoietic cells at day 0 and after 48-h culture. CD117 (SCF receptor) expression, although very heterogeneous according to the subpopulations and the sources of progenitors evaluated, seems not to correlate with the difference of progenitor cell sensitivity to SCF nor with their proliferative capacity. Considering the importance of the c-kit/SCF complex in the adhesion of stem cells to the microenvironment, several observations are relevant. The density of CD117 antigen expression (expressed in terms of mean equivalent soluble fluorescence, MESF) is significantly lower on fresh PB cells than on their BM (P < 0.017) and CB (P < 0.004) counterparts, particularly in the immature CD34+CD38- population (560 +/- 131, 2121 +/- 416 and 1192 +/- 129 MESF respectively); moreover, when PB and BM CD34+CD38- cells are stimulated for 48 h with SCF + IL-3, the CD117 expression decreases by 1.5- and 1.66-fold, respectively. This reduction could modify the functional capacities of ex vivo PB and BM manipulated immature progenitor cells.     

Autologous CD34+ cells transplantation after FAMP treatment in a patient with CLL and persisting AIHA: complete remission of lymphoma with control of autoimmune complications.

A 48-year-old male with CLL and concomitant AIHA unresponsive to chlorambucil was treated with fludarabine. The remission of CLL and improvement of the AIHA was achieved, but the patient remained steroid dependent. Therefore, high-dose chemotherapy followed by CD34-selected autologous peripheral blood stem cells transplantation was performed and this led to long-term clinical, immunophenotypic and molecular remission with disappearance of AIHA. Twenty-three months later, the CLL recurred with signs of AIHA. In this patient with AIHA, HDC and selected CD34+ cells completely, though temporarily, controlled both CLL and associated immune complications. This case illustrates the potential application of this approach in the management of CLL patients with immune complications.     

Circulation of CD34+ hematopoietic stem cells in the peripheral blood of high-dose cyclophosphamide-treated patients: enhancement by intravenous recombinant human granulocyte-macrophage colony-stimulating factor

We report that hematopoietic progenitor cells expressing the CD34 antigen (CD34+ cells) transiently circulate in the peripheral blood (PB) of cancer patients treated with 7 g/m2 cyclophosphamide (HD-CTX) with or without recombinant human granulocyte macrophage-colony stimulating factor (rHuGM-CSF). In adult humans, CD34+ cells represent a minor fraction (1% to 4%) of bone marrow (BM) cells, comprising virtually all hematopoietic colony-forming progenitors in vitro and probably also stem cells capable of restoring hematopoiesis of lethally irradiated hosts. We show that CD34+ cell circulation is fivefold enhanced by rHuGM-CSF 5.5 protein micrograms/kg/day by continuous intravenous infusion for 14 days after HD-CTX. During the third week after HD-CTX (ie, when CD34+ cells peak in the circulation), large-scale collection of PB leukocytes by three to four continuous-flow leukaphereses allows the yield of 2.19 to 2.73 x 10(9) or 0.45 to 0.56 x 10(9) CD34+ cells depending on whether or not patients receive rHuGM-CSF. The number of CD34+ cells retrieved from the circulation by leukaphereses exceeds the number that can be harvested by multiple BM aspirations under general anesthesia. Thus, after therapy with HD-CTX and rHuGM-CSF, PB represents a rich source of hematopoietic progenitors possibly usable for restoring hematopoiesis after myeloablative chemoradiotherapy. To determine whether CD34+ cells found in the PB are equivalent to their marrow counterpart, we evaluated their in vitro growth characteristics and immunological phenotype by colony assays and dual-color immunofluorescence, respectively. We show that PB CD34+ cells possess qualitatively normal hematopoietic colony growth and high cloning efficiency comparable to that observed with BM CD34+ cells. In addition, PB CD34+ cells display heterogeneous surface membrane differentiation antigens analogous to BM CD34+ cells. The availability of large quantities of CD34+ cells by leukapheresis is relevant to the field of stem cell transplantation and possibly to genetic manipulations of the hematopoietic system in humans.     

Phenotypic and functional analysis of bone marrow progenitor cell compartment in bone marrow failure

SUMMARY. Many laboratory findings have demonstrated that the haemopoietic stem cell compartment is defective in aplastic anaemia (AA). AA bone marrow (BM) and peripheral blood (PB) are profoundly deficient in colonyforming cells, and AA progenitors fail to proliferate in longterm assays even in the presence of an intact stroma. Our study was designed to characterize some quantitative and qualitative aspects of the progenitor cell defect in AA. Using flow cytometric analysis of BM from new AA patients and from those recovering after immunosuppressive therapy, we determined that the numbers of CD34⁺ and CD33⁺ cells were markedly decreased in AA. Although PB neutrophil counts did not correlate with BM CD34⁺ cell numbers in acute disease, there was an association between the overall severity of the disease and the degree of CD34⁺ cell reduction. A decrease in BM CD33⁺ cells was a common finding in MDS patients, but reduction in CD34⁺ cells was found only in some hypoplastic MDS cases. Sorting experiments demonstrated lower plating efficiency for purged CD34⁺ cells from AA BM in comparison to controls. Thus, diminished colony formation of total BM appeared to result from both quantitative and qualitative defects. Based on the association between increased cycling and c-kit receptor expression on CD34⁺ cells, we found that the mitotically active CD34⁺ cells bearing the c-kit antigen were reduced in AA. With clinical improvement, CD34⁺ and CD33⁺ cells increased in correlation with PB parameters, but they did not return to normal values. Sorted CD34⁺ cells from recovered patents showed improved plating efficiency. In patients with aplastic anaemia, use of CD34 antigen as a phenotypic marker of progenitor cells may be helpful for the analysis of the early haemopoietic cell compartment and BM recovery.     

HIGH-DOSE THERAPY WITH PERIPHERAL BLOOD STEM CELL TRANSPLANTATION RESULTS IN A SIGNIFICANT REDUCTION OF THE HaEMOPOIETIC PROGENITOR CELL COMPARTMENT

In order to study the effect of high-dose therapy with peripheral blood stem cell transplantation (PBSCT) on the haemopoietic reserve in man, the number and composition of bone marrow (BM) and peripheral blood (PB)-derived progenitor cells were examined in 137 cancer patients. In 45 patients, paired samples from BM and PB were obtained before PBSC mobilization and 6–27 months after transplantation. Following PBSCT, the proportion of CD34⁺ cells was significantly smaller than before mobilization (BM 1.99±0.24 versus 0.8±0.09, P <0.001), and no change was observed at several follow-up visits thereafter.
The reduction was most pronounced for the primitive BM progenitor subsets such as the CD34⁺/DR⁻ and CD34⁺/Thy-1⁺ cells. The impairment of hematopoiesis was also reflected by a significant reduction in the plating efficiency of BM and PB samples.
No relationship was found between the decrease in the proportion of CD34⁺ cells and any particular patient characteristics, kind of high-dose therapy or the CD34⁺ cell content in the autograft.
In conclusion, high-dose therapy with PBSC transplantation is associated with a long-term impairment of the haemopoietic system. The reduction in the number of haemopoietic progenitor cells is not associated with a functional deficit, as peripheral blood counts post-transplantation were normal in the majority of patients.     

Mobilization of peripheral blood stem cells in CLL patients after front-line fludarabine treatment

Autologous peripheral blood stem cell transplantation is performed in an increasing number of chronic lymphocytic leukaemia (CLL) patients who are in the first remission following fludarabine treatment. There are contradictory data about the adverse impact of fludarabine on stem cell harvest. We analysed retrospectively mobilization results in 56 poor-risk CLL patients (median age: 56 years) who underwent first-line treatment with fludarabine and cyclophosphamide. The mobilization, consisting of cyclophosphamide 3 g/m(2) and granulocyte colony-stimulating factor (G-CSF) 10 microg/kg per day, was performed with a median of 77 days following the last fludarabine course. The target yield was >or=2.0x10(6) CD34+ cells/kg. The procedure was successful in 23 (41%) patients. A median of 3.3x10(6) CD34+ cells/kg was collected per patient. The successful mobilization was associated with a longer interval from the last chemotherapy (>2 months). The mobilization result was not influenced by the number of fludarabine cycles. No correlation was found in other parameters such as disease stage at diagnosis, disease status at stimulation or age. The poorly mobilized patients had significantly lower prestimulation blood counts (platelets, WBC and haemoglobin). Our data show that fludarabine does not generally prevent the stem cell mobilization; nevertheless, mechanisms related to the impact of fludarabine on stem cell harvest must be further investigated.     

Identification and comparison of CD34-positive cells and their subpopulations from normal peripheral blood and bone marrow using multicolor flow cytometry.

Four-color flow cytometry was used with a cocktail of antibodies to identify and isolate CD34+ hematopoietic progenitors from normal human peripheral blood (PB) and bone marrow (BM). Mature cells that did not contain colony forming cells were resolved from immature cells using antibodies for T lymphocytes (CD3), B lymphocytes (CD20), monocytes (CD14), and granulocytes (CD11b). Immature cells were subdivided based on the expression of antigens found on hematopoietic progenitors (CD34, HLA-DR, CD33, CD19, CD45, CD71, CD10, and CD7). CD34+ cells were present in the circulation in about one-tenth the concentration of BM (0.2% v 1.8%) and had a different spectrum of antigen expression. A higher proportion of PB-CD34+ cells expressed the CD33 myeloid antigen (84% v 43%) and expressed higher levels of the pan leukocyte antigen CD45 than BM-CD34+ cells. Only a small fraction of PB-CD34+ cells expressed CD71 (transferrin receptors) (17%) while 94% of BM-CD34+ expressed CD71+. The proportion of PB-CD34+ cells expressing the B-cell antigens CD19 (10%) and CD10 (3%) was not significantly different from BM-CD34+ cells (14% and 17%, respectively). Few CD34+ cells in BM (2.7%) or PB (7%) expressed the T-cell antigen CD7. CD34+ cells were found to be predominantly HLA-DR+, with a wide range of intensity. These studies show that CD34+ cells and their subsets can be identified in normal PB and that the relative frequency of these cells and their subpopulations differs in PB versus BM.     

Peripheral blood progenitor cell (PBPC) counts during steady-state hematopoiesis allow to estimate the yield of mobilized PBPC after filgrastim (R-metHuG-CSF)-supported cytotoxic chemotherapy [see comments]

Peripheral blood progenitor cells (PBPC) can be mobilized using cytotoxic chemotherapy and cytokines. There is a substantial variability in the yield of hematopoietic progenitor cells between patients. We were looking for predictive parameters indicating a patient's response to a given mobilization regimen. Multiparameter flow- cytometry analysis and clonogenic assays were used to examine the hematopoietic progenitor cells in bone marrow (BM) and peripheral blood (PB) before filgrastim (R-metHuG-CSF; Amgen, Thousand Oaks, CA)- supported chemotherapy and in PB and leukapheresis products (LPs) in the recovery phase. Fifteen patients (four with high-grade non- Hodgkin's lymphoma [NHL], two with low-grade NHL, two with Hodgkin's disease, two with multiple myeloma, three with breast cancer, one with ovarian cancer, and one with germ cell tumor) were included in this study. The comparison of immunofluorescence plots showed a homogenous population of strongly CD34+ cells in steady-state and mobilized PB whereas in steady-state BM, the CD34+ cells ranged from strongly positive with continuous transition to the CD34- population. Consistent with the similarity in CD34 antigen expression, a correlation analysis showed steady-state PB CD34+ cells (r = .81, P < .001) and colony- forming cells (CFCs; r = .69, P < .01) to be a measure of a patient's mobilizable CD34+ cell pool. Individual estimates of progenitor cell yields could be calculated. With a probability of 95%, eg, 0.4 steady- state PB CD34+ cells x 10(6)/L allowed to collect in six LPs 2.5 x 10(6) CD34+ cells/kg, the reported threshold-dose of progenitor cells required for rapid and sustained engraftment after high-dose therapy. For the total steady-state BM CD34+ cell population, a weak correlation (r = .57, P < .05) with the mobilized CD34+ cells only became apparent when an outlier was removed from the analysis. Neither the CD34+ immunologic subgroups defined by the coexpression of the myeloid lineage-associated antigens CD33 or CD45-RA or the phenotypically primitive CD34+/HLA-DR- subset nor the BM CFC count had a predictive value for the mobilization outcome. This may be caused by the additional presence of maturing progenitor cells in BM, which express lower levels of the CD34 antigen and do not circulate. Our results permit us to recognize patients who are at risk to collect low numbers of progenitor cells and those who are likely to achieve sufficient or high progenitor cell yields even before mobilization chemotherapy is administered.     

Mobilization of peripheral blood progenitor cells (PBPC) in patients undergoing chemotherapy followed by autologous peripheral blood stem cell transplant (SCT) for high risk breast cancer (HRBC).

We have determined the effect of delayed addition of G-CSF after chemotherapy on PBPC mobilization in a group of 30 patients with high risk breast cancer (HRBC) undergoing standard chemotherapy followed by high-dose chemotherapy (HDCT) and autologous SCT. Patients received FAC chemotherapy every 21 days followed by G-CSF at doses of 5 microg/kg/day starting on day +15 (groups 1 and 2) or +8 (group 3) after chemotherapy. PBPC collections were performed daily starting after 4 doses of G-CSF and continued until more than 2.5 x 10(6) CD34+ cells had been collected. In group 1, steady-state BM progenitors were also harvested and used for SCT. Groups 2 and 3 received PBPC only. The median number of collections was three in each group. Significantly more PB CD34+ cells were collected in patients receiving G-CSF starting on day 8 vs day 15 (9.43 x 10(6)/kg and 6.2 x 10(6)/kg, respectively) (P < 0.05). After conditioning chemotherapy all harvested cells including BM and PBPC were reinfused. Neutrophil and platelet engraftment was significantly faster in patients transplanted with day 8 G-CSF-mobilized PBPC (P < 0.05) and was associated with lower transplant related morbidity as reflected by days of fever, antibiotics or hospitalization (P < 0.05). Both schedules of mobilization provided successful long-term engraftment with 1 year post-transplant counts above 80% of pretransplant values. In conclusion, we demonstrate that delayed addition of G-CSF results in successful mobilization and collection of PBPC with significant advantage of day 8 G-CSF vs day 15. PBPC collections can be scheduled on a fixed day instead of being guided by the PB counts which provides a practical advantage. Transplantation of such progenitors results in rapid short-term and long-term trilineage engraftment.     

Kinetics of engraftment of CD34(-) and CD34(+) cells from mobilized blood differs from that of CD34(-) and CD34(+) cells from bone marrow

OBJECTIVE: Mobilized peripheral blood (PB) progenitors are increasingly used in autologous and allogeneic transplantation. However, the short- and long-term engraftment potential of mobilized PB or bone marrow (BM) has not been directly compared. Although several studies showed that BM-derived Lin(-)CD34(-) cells contain hemopoietic progenitors, no studies have addressed whether Lin(-)CD34(-) cells from mobilized PB contain hemopoietic progenitors. Here, we compared the short- and long-term engraftment potential of CD34(+) cells and Lin(-)CD34(-) cells in BM and PB of normal donors who received 5 days of granulocyte colony-stimulating factor (G-CSF). MATERIALS AND METHODS: 35 x 10(3) CD34(+) or Lin(-)CD34(-) cells from G-CSF mobilized BM and PB of normal donors were transplanted in 60-day-old fetal sheep. Animals were evaluated 2 and 6 months after transplantation for human hemopoietic cells. In addition, cells recovered after 2 months from fetal sheep were serially passaged to secondary and tertiary recipients to assess long-term engrafting cells. RESULTS: Mobilized PB CD34(+) cells supported earlier development of human hemopoiesis than BM CD34(+) cells. When serially transferred to secondary and tertiary recipients, earlier exhaustion of human hematopoiesis was seen for PB than BM CD34(+) cells. A similar degree of chimerism was seen for Lin(-)CD34(-) cells from PB or BM in primary recipients. We again observed earlier exhaustion of human hemopoiesis with serial transplantation of PB than BM Lin(-)CD34(-) cells. CONCLUSIONS: Differences exist in the short- and long-term repopulating ability of cells in PB and BM from G-CSF mobilized normal donors, and this is independent of the phenotype. Studies are ongoing to examine if this reflects intrinsic differences in the repopulating potential between progenitors from PB and BM, or a lower frequency of long-term repopulating cells in PB than BM CD34(+) and Lin(-)CD34(-) cells, that may not be apparent if larger numbers of cells are transplanted.     

Hematologic effects of flt3 ligand in vivo in mice

We have investigated the effects of in vivo treatment with flt3 ligand (FL) on murine hematopoiesis, including mobilization of progenitors into the peripheral blood (PB). Mice were injected once daily with 10 micrograms recombinant human FL for 15 days. On days 3, 5, 8, 10, 15, and 22, mice were killed and analyzed for the number of leukocytes and colony-forming units (CFU) in bone marrow (BM), spleen, and PB. Splenic and PB cellularity increased with time in FL-treated mice. In the spleen, there was an increase in B cells, myeloid cells, and nucleated erythroid cells; in the PB, there was an increase in lymphocytes, granulocytes, and monocytic cells. The maximal number of CFU in the BM was observed after 3 days of FL treatment, giving 3.7- and 7.3-fold increases in CFU-granulocyte-macrophage (CFU-GM) and CFU-granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM), respectively, compared with mouse serum albumin (MSA)-treated controls. After 8 days of FL treatment, there was a maximal 123- and 108-fold increase in splenic CFU-GM and CFU-GEMM, respectively. The maximal number CFU-GM and CFU- GEMM were seen in PB on day 10, with 537- and 585-fold increases, respectively. Burst-forming units-erythroid (BFU-E) increased in the same time frame as those of CFU-GM and CFU-GEMM in BM, spleen, and PB, although the magnitude was not as great. Primitive day-13 CFU-spleen (CFU-S) and phenotypically defined stem cells were also mobilized into the PB of FL-treated mice with similar kinetics and magnitude to that of CFU-GM and CFU-GEMM. We conclude from these studies that FL, when administered as a single agent, is a potent mobilizer of hematopoietic progenitors into the PB.