Cord blood G(0) CD34+ cells have a thousand-fold higher capacity for generating progenitors in vitro than G(1) CD34+ cells.

We examined the functional differences between G(0) and G(1) cord blood CD34+ cells for up to 24 weeks in serum-free suspension culture, containing Flt-3 ligand, thrombopoietin and stem cell factor. By week 24, there is more than a 1,000-fold difference in granulocyte, macrophage-colony-forming cells (GM-CFC) cumulative production between the two populations, with cultures initiated from G(0) demonstrating an amplification of 1.1 x 10(5)-1.8 x 10(6) of GM-CFC compared to 45-2.7 x 10(3) for the G(1) cells. Cells from the initial G(0) population are able to produce about 250-fold higher numbers of BFU-E than those from G(1) which translates to 3 x 10(3)-1.1 x 10(5)-fold expansion and 25-390-fold expansion for G(0) and G(1), respectively. This amplification of the progenitor cells is reflected in finding that a greater proportion of the progeny of the G(0) population are CD34+, resulting in a 600-fold expansion of CD34+ cells at week 8. As in other in vitro systems, total cell expansion is less discriminatory of stem cell behavior than progenitor cells, and there is no significant difference in total cell numbers between G(0) and G(1) cultures with a mean fold expansion of 2 x 10(7) at 24 weeks.

Accelerated telomere shortening following allogeneic transplantation is independent of the cell source and occurs within the first year post transplant.

Telomere shortening has been documented in the blood cells of recipients of allogeneic bone marrow transplants compared with their donors. Allogeneic peripheral blood progenitor cells (PBPCs) have been increasingly used as an alternative to bone marrow. Their advantages include earlier engraftment and immune reconstitution following transplantation. We have measured telomere length of neutrophils and T cells in fully engrafted recipients of allogeneic bone marrow (n = 19) and allogeneic PBPC (n = 17) and also measured sequential telomere length in four patients after transplantation. Overall, significant telomere shortening occurred in recipients in neutrophils (0.3 kb, P < 0.001) and T cells (0.2 kb, P = 0.045). The data demonstrate that first, the degree of shortening was the same for BM and PBPC transplants and was not related to the time taken to engraft neutrophils and platelets and second, telomere shortening occurs in the first year post transplant without further shortening during the period of observation. These data suggest that the superiority of engraftment seen in PBPC transplants is independent of telomere shortening and other mechanisms such as homing or seeding may be more important.

CCR1 chemokine receptor expression isolates erythroid from granulocyte-macrophage progenitors.

Simple methods that separate progenitor cells of different hemopoietic lineages would facilitate studies on lineage commitment and differentiation. We used an antibody specific for the chemokine receptor CCR1 to examine mononuclear cells isolated from cord blood samples. When CD34(+) cells were separated into CD34(+)CCR1(+) and CD34(+)CCR1(-) cells and plated in colony-forming assays, the granulocyte/macrophage progenitors were found almost exclusively in the CD34(+)CCR1(+) cells. In contrast, the CD34(+)CCR1(-) cells contained the majority of the erythroid progenitors. There was a highly significant difference (P<0.002) in the total percentage distribution of both granulocyte-macrophage colony-forming cells and erythroid burst-forming units between the two populations. This is the first report of separation of erythroid progenitors from granulocyte/macrophage progenitors using a chemokine receptor antibody in cord blood samples. These results suggest that at the clonogenic progenitor cell stage the expression of CCR1 might be lineage-specific. This method should prove useful for studies on erythroid progenitor and granulocyte/macrophage differentiation.

NOD/SCID repopulating cells but not LTC-IC are enriched in human CD34+ cells expressing the CCR1 chemokine receptor.

Human haemopoietic stem and progenitor cells may be distinguished by the pattern of cell surface markers they display. The cells defined as 'stem' cells are heterogeneous and lack specific markers for their detection. However, they may be identified in in vitro assays such as the long-term culture initiating cell (LTC-IC) and in transplant assays involving immunosuppressed NOD/SCID mice. It is still not clear to what extent, if any, these cell populations overlap. The chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) prolongs survival of LTC-IC in suspension cultures and we now show that in longterm bone marrow cultures (LTBMC) maintenance of haemopoiesis was significantly better from the CD34+ cells which possess MIP-1alpha receptors (P < 0.006). We examined one MIP-1alpha receptor, CCR1, which is present on CD34+ cells from haemopoietic tissues. In LTBMC the production of GM-CFC from CD34+CCR1- cells was significantly higher (P < 0.02) than that from CD34+CCR1+ cultures and the incidence of LTC-IC was 3- to 6-fold higher in the CD34+CCR1- cell fraction. In contrast, the cells responsible for high levels of engraftment in NOD/SCID mice were contained in the CD34+CCR1+ cell fraction. The CD34+CCR1+ cells engrafted to high levels in NOD/SCID and generated large numbers of progenitor cells. Therefore, we conclude that LTC-IC and SRC may be distinguished on the basis of expression of the chemokine receptor CCR1.

Interest of cord blood stem cells.

Interest in cord blood stem cells was raised because of the possibility, now realised, of their use in clinical transplantation. The availability of only limited numbers of stem cells in cord blood compared to bone marrow or peripheral blood apheresis after cell mobilisation, led to experimental approaches that first aimed to characterise and then manipulate the stem cells present in cord blood. Their phenotypical and functional characteristics are not identical to those of stem cells in the bone marrow or those cells mobilised into the circulation. The cells selected for phenotype plus Go status show the higher capacity to generate progenitor cells in vitro and will offer the opportunity for mechanistic studies of stem cell self-renewal and proliferation. Another important field of exploration is to investigate the capacity of stem cells in cord blood for differentiation to tissues other than haemopoietic and to establish whether haemopoietic and non-haemopoietic lineages originate in truly multipotential cells or in cells coexisting in cord blood, which have already been limited to differentiation into specific tissue.

Interactions of human prostatic epithelial cells with bone marrow endothelium: binding and invasion.

Prostate cancer shows a propensity to form secondary tumours within the bone marrow. Such tumours are the major cause of mortality in this disease. We have developed an in vitro system to study the binding of prostate epithelial cells to bone marrow endothelium (BME) and stroma (BMS). The metastatic prostate cancer cell line, PC3 (derived from a bone metastasis), was seeded onto confluent layers of BME and its binding characteristics compared to human umbilical vein endothelial cells (HUVEC), lung endothelium (Hs888Lu) and BMS. The PC3 cell line showed significantly increased binding to BME (P< 0.05) compared to endothelium derived from HUVEC and lung or BMS with maximal binding occurring at 1 h. Following pre-incubation with a beta1 integrin antibody PC3 binding to BME was inhibited by 64% (P< 0.001). Antibodies directed against the integrins beta4, alpha2, alpha4, alpha5 and the cellular adhesion molecules P-selectin, CD31, VCAM-1 and sialy Lewis X showed no effect on blocking PC3 binding. Primary prostatic epithelial cells from both malignant (n = 11) and non-malignant tissue (n = 11) also demonstrated equivalent levels of increased adhesion to BME and BMS compared to HUVEC, peaking at 24 h. Further studies examined the invasive ability of prostate epithelial cells in response to bone marrow endothelium using Matrigel invasion chamber assays. In contrast to the previous results, malignant cells showed an increase (1000 fold) in invasive ability, whilst non-malignant prostate epithelia did not respond. We have shown that both malignant and non-malignant prostate epithelial cells can bind at equivalent levels and preferentially to primary human bone marrow endothelium in comparison to controls. However, only malignant prostate epithelia show increased invasive ability in response to BME.

Dynamics of telomere shortening in neutrophils and T lymphocytes during ageing and the relationship to skewed X chromosome inactivation patterns.

Human haemopoiesis undergoes profound changes throughout life, resulting in compromised regenerative capacity of haemopoietic stem cells. It has been suggested that telomere shortening results in senescence of haemopoietic stem cell subsets and may influence the balance between stem cell renewal and proliferation. Telomere length and telomerase activity was measured in whole blood leucocytes, neutrophils and T cells from cord blood and individuals aged from 1 year to 96 years. Rapid telomere shortening [700 base pairs (bp)] was demonstrated in the first year of life, followed by a gradual decline of 31 bp/year. T cells were shown to have longer telomeres than neutrophils (mean difference 372 bp, P = < 0.001) but demonstrated similar rates of shortening (20 +/- 0.3 bp/year vs. 22 +/- 0.3 bp/year). Telomerase was detectable in T cells but not in neutrophils, suggesting that telomerase is not the rate-limiting step for regulation of telomere length in haemopoietic cells. Stem cell utilization as measured by X chromosome inactivation patterns was found to be independent of telomere length. This supports the concept that age-dependent skewed haemopoiesis is the result of random stem cell loss or X-allelic exclusion rather than telomeric senescence. These studies provide insight into the ageing process and a reference point for evaluating replicative stress in individuals of different age groups.

Detection of hTERT protein by flow cytometry.

Telomerase is a telomere-specific DNA polymerase consisting of protein and RNA components, which is activated in germline cells and the majority of cancers and serves to counter the consequences of telomere shortening. The protein component, hTERT, is believed to be the catalytic subunit of human telomerase and its expression at the mRNA level correlates well with telomerase activity in vitro. Current techniques for assaying telomerase activity detect only the mean activity in a sample and are unable to isolate specific cell sub-populations. This report describes the development and validation of a cellular, immunofluorescence-based flow cytometry assay that allows detection of intranuclear hTERT while maintaining identifiable cell population characteristics. The assay was shown to be both sensitive to changes in telomerase expression and was semi-quantitative. In both cell line differentiation experiments and in primary cells, a good correlation existed between hTERT expression measured by flow cytometry and telomerase activity detected by the telomeric repeat amplification protocol (TRAP). The method developed offers a quick, simple and reproducible cellular-based assay for hTERT expression. This assay will provide a useful, new tool for future investigations, facilitating the analysis of hTERT expression in mixed cell populations.

Characterisation of the differential response of normal and CML haemopoietic progenitor cells to macrophage inflammatory protein-1alpha.

The clonogenic cells of chronic myeloid leukaemia (CML), unlike normal haemopoietic colony forming cells (CFC), are resistant to the growth inhibitory effects of the chemokine, macrophage inflammatory protein-1alpha (MIP-1alpha). Here, we tested the hypothesis that MIP-1alpha protects normal, but not CML, CFC from the cytotoxic effects of the cell-cycle active drug cytosine arabinoside (Ara-C). Using a 24-h Ara-C protection assay we showed that MIP-1alpha confers protection to normal CFC but also sensitizes CML CFC to Ara-C. The differential MIP-1alpha responsiveness was not due to a down-regulation of MIP-1alpha receptors on CML CD34+ cells as flow cytometric analysis showed similar binding of a biotinylated MIP-1alpha molecule to normal and CML CD34+ cells. Flow cytometric analysis of the MIP-1alpha receptor subtype CCR-5 revealed comparable CCR-5 expression levels on normal and CML CD34+ cells. Furthermore, culture of CD34+ cells for 10 h in the presence of TNF-alpha resulted in an increased MIP-1alpha receptor expression on both normal and CML CD34+ cells. Our data suggest that the unresponsiveness of CML CFC to the growth inhibitory effect of MIP-1alpha is not caused by a lack of MIP-1alpha receptor or total uncoupling of the MIP-1alpha responsiveness but may be due to an intracellular signalling defect downstream of the receptors.

Scatter factor influences the formation of prostate epithelial cell colonies on bone marrow stroma in vitro.

Prostate cancer metastases form selectively in the bone marrow. Previously we demonstrated motility was important for the formation of primary prostatic epithelial cell colonies in bone marrow stroma (BMS) co-culture. In this study we looked at the influence of motility factors on the colony formation of epithelial cells derived from benign (bPEC) or malignant (mPEC) prostate tissue. After 7 days co-culture we found that anti-scatter factor consistently inhibited prostate epithelial cell colony formation on BMS (7/7 mPEC and 4/7 bPEC samples showed significant inhibition). Antibodies against bFGF and 5T4 did not significantly affect colony formation. Addition of fibroblast conditioned media (derived from benign prostates) to co-cultures stimulated the colony formation of bPEC (170%) and mPEC (252%). This stimulation was eliminated by depletion of SF from the conditioned media. Immunohistochemical staining found c-Met expression in 5/6 bPEC cultures and 7/9 mPEC cultures. When grown in BMS co-culture expression of c-Met was positive in 3/6 bPEC and 2/7 mPEC samples. In conclusion, scatter factor influences the in vitro formation of prostate epithelial cell colonies on BMS co-culture.

Multicentre European study comparing selection techniques for the isolation of CD34+ cells.

Primitive haemopoietic cells are required for studies in both the clinical and research fields and a number of systems have been developed to facilitate isolation of these haemopoietic cell populations. We have analysed the results from several European centres using positive selection of CD34+ cells from haemopoietic tissues (n = 110). Four selection techniques including immunoaffinity columns (Ceprate LC), immunomagnetic beads (Dynabeads, Baxter Isolex 50) and submicroscopic magnetic beads (MACS) were used and the selected CD34+ cells were assessed for purity, yield and enrichment of colony-forming cells (CFC). The mean purities for all samples ranged from 68.4-78.4% for MACS, 33.9-69.9% for Dynabeads, 46.9-66.8% for Ceprate LC and 43.2-65% for Baxter Isolex 50. Yields were variable with all techniques. On average CFC enrichment using the immunoaffinity columns was greater than that observed for the other systems. Some techniques appear to be problematic and may require further expertise to improve the results. Nevertheless, the study demonstrates that highly purified CD34+ cells can be isolated from various haemopoietic sources, though yield and CFC enrichment varies significantly depending on the technique selected. This extends our previous report indicating that not all selection methods generate similar results and that there are differences in the purity, number and colony-forming ability of the cells recovered.

Distinct biological effects of macrophage inflammatory protein-1alpha and stroma-derived factor-1alpha on CD34+ hemopoietic cells.

Chemokines are important regulators of both hemopoietic progenitor cell (HPC) proliferation and adhesion to extracellular matrix molecules. Here, we compared the biological effects of the CC chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) with those of the CXC chemokine stroma-derived factor-1alpha (SDF-1alpha) on immunomagnetically purified CD34+ cells from leukapheresis products (LP CD34+). In particular, studies on chemokine-induced alterations of LP CD34+ cell attachment to fibronectin-coated plastic surfaces, proliferation of these cells in colony-forming cell (CFC) assays and intracellular calcium mobilization were performed. MIP-1alpha but not SDF-1alpha was found to increase the adhesion of LP CD34+ cells to fibronectin in a dose-dependent manner. Both chemokines elicited growth-suppressive effects on LP CD34+ cells in CFC assays. While MIP-1alpha reduced the number of granulomonocytic (CFC-GM) and erythroid (BFU-E) colonies to the same extent, SDF-1alpha showed a significantly greater inhibitory effect on CFC-GM than BFU-E. Finally, we demonstrated that SDF-1alpha but not MIP-1alpha triggers increases in intracellular calcium in LP CD34+ cells. The SDF-1alpha-induced calcium response was rapid and concentration-dependent, with a maximal stimulation observed at > or = 15 ng/ml. In conclusion, our data suggest distinct biological properties of SDF-1alpha and MIP-1alpha in terms of modulation of LP CD34+ cell adhesion to fibronectin and intracellular calcium levels. However, comparable growth-suppressive effects on HPC proliferation were observed, indicating that this feature may be independent of chemokine-induced calcium responses.

Content of long-term culture-initiating cells, clonogenic progenitors and CD34 cells in apheresis harvests of normal donors for allogeneic transplantation, and in patients with acute myeloid leukaemia or multiple myeloma.

Using a limiting dilution assay the frequency of long-term culture-initiating cells (LTC-IC) in the apheresis products following mobilization by granulocyte-colony stimulating factor (G-CSP) with or without chemotherapy from 14 normal donors (ND) for allogeneic bone marrow transplantation, 16 patients with multiple myeloma (MM) and 15 patients with acute myeloid leukaemia (AML), where the aphereses were intended for autologous transplantation, were compared. The estimated median incidences of LTC-IC in the first apheresis products from ND, MM and AML were 1/3289, 1/1775 and 1/13075 mononuclear cells (MNC) respectively. The patients with AML had a significantly lower incidence compared with the other two groups (P < 0.0001). There was a positive correlation between the incidence of LTC-IC and the number of CD34+ cells, the number of GM-CFC, and the number of BFU-E. The positive association with GM-CFC or BFU-E was weaker. In these experiments the percentage of CD34+ cells was the best predictor for the frequency of LTC-IC in the peripheral blood progenitor cells (PBPC). In eight cases of MM the LTC-IC assay was performed for both the first and second harvest. All cases had a lower LTC-IC frequency in the second harvest compared with the first, an average of 23% (13-42%, 95% confidence interval) and this reduction was statistically significant (P<0 001); CD34+ cells were also lower (P< 0.001).

Properties of peripheral blood and cord blood stem cells.

Mobilized peripheral blood and cord blood are used for transplantation in adults and children. Currently methods which assess the engraftment potential of these cells rely on nucleated cell count, clonogenic colony assays (GM-CFC) and CD34+ cell enumeration. However, data have accumulated which indicate that the cells responsible for short-term and long-term engraftment are different and may be identified by a variety of techniques, including immunophenotyping, in vitro and in vivo assays. There is also evidence that primitive cells in peripheral blood progenitor cell grafts and cord blood are heterogeneous, as cells with similar functional behaviour express different phenotypes. Despite intensive research, the isolation and identification of a homogeneous population of human stem cells is still elusive. Nevertheless, it is possible to obtain CD34+ subpopulations enriched in primitive cells with many of the properties expected of stem cells. Using these cell fractions, the cytokines that induce proliferation, amplification, differentiation and self-renewal are being defined in order to develop improved protocols for expansion of specific populations. From these studies a number of interesting facts have emerged. Certain growth factors frequently used for progenitor cell expansion and gene transduction studies also induce differentiation and impair long-term engraftment. Further, the cytokines required for progenitor cell expansion are probably different to those which favour expansion of the primitive cells, with both the cell cycle status of CD34+ cells as well as the implication of telomere shortening probably needing to be considered where ex vivo manipulation is contemplated.

CD34+AC133+ cells isolated from cord blood are highly enriched in long-term culture-initiating cells, NOD/SCID-repopulating cells and dendritic cell progenitors.

The AC133 antigen is a novel antigen selectively expressed on a subset of CD34+ cells in human fetal liver, bone marrow, and blood as demonstrated by flow cytometric analyses. In this study, we have further assessed the expression of AC133 on CD34+ cells in hemopoietic samples and found that there was a highly significant difference between normal bone marrow and cord blood versus aphereses (p <0.0001) but not between bone marrow and cord blood. Most of the clonogenic cells (67%) were contained in the CD34+AC133+ fraction. Compared with cultures of the CD34+AC133- cells, generation of progenitor cells in long-term culture on bone marrow stroma was consistently 10- to 100-fold higher in cultures initiated with CD34+AC133+ cells and was maintained for the 8-10 weeks of culture. Only the CD34+AC133+ cells were capable of repopulating NOD/SCID mice. Human cells were detectable as early as day 20, with increased levels (67%) apparent 40 days post-transplantation. Five thousand CD34+AC133+ cells engrafted about 20% of the mice, while no engraftment was observed in animals transplanted with up to 1.2 x 10(5) CD34+AC133- cells. The CD34+AC133+ population was also enriched (seven-fold) in dendritic cell precursors, and the dendritic cells generated were functionally active in a mixed lymphocyte reaction assay. AC133+ cells should be useful in the study of cellular and molecular mechanisms regulating primitive hemopoietic cells.

Expression of macrophage inflammatory protein-1 receptors in human CD34(+) hematopoietic cells and their modulation by tumor necrosis factor-alpha and interferon-gamma.

Macrophage inflammatory protein-1alpha (MIP-1alpha) can stimulate growth inhibitory and potent chemotactic functions in hematopoietic cells. To investigate whether the action of MIP-1alpha may be regulated at the cellular receptor level, we studied the expression and modulation of MIP-1alpha receptors on CD34(+) cells isolated from normal bone marrow (NBM), umbilical cord blood (CB), and leukapheresis products (LP). Expression of MIP-1alpha receptors on CD34(+) cells was analyzed by two-color flow cytometry using a biotinylated MIP-1alpha molecule. The mean percentage of LP CD34(+) cells expressing the MIP-1alpha receptors was 67.7 +/- 7.2% (mean +/- SEM; n = 22) as compared with 89.9 +/- 2.6% (n = 10) and 74.69 +/- 7.04% (n = 10) in CB and NBM, respectively (P = .4). The expression of the MIP-1alpha receptor subtypes on LP CD34(+) cells was studied by indirect immunofluorescence using specific antibodies for the detection of CCR-1, CCR-4, and CCR-5. Microscopical examination revealed a characteristic staining of the cytoplasmic cell membrane for all three receptor subtypes. Detailed analysis of two LP samples showed that 65.8%, 4.4%, and 30.5% of CD34(+) cells express CCR-1, CCR-4, and CCR-5, respectively. Culture of LP CD34(+) cells for 24 to 36 hours in the presence of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) resulted in a significant increase in MIP-1alpha receptor expression. TNF-alpha induced MIP-1alpha receptor upregulation in a time- and concentration-dependent manner. Our results suggest that inhibitory cytokines produced by the bone marrow microenvironment are likely to be involved in the regulation of MIP-1alpha receptor expression on hematopoietic cells.

Differential response of CD34+ cells isolated from cord blood and bone marrow to MIP-1 alpha and the expression of MIP-1 alpha receptors on these immature cells.

Macrophage inflammatory protein-1 alpha (MIP-1alpha) has been shown to have a role in the control of myeloid stem and progenitor cell proliferation. Recent evidence suggests that MIP-1alpha also has a stimulatory effect on proliferation of mature progenitors as well as an inhibitory effect on immature progenitors in vitro. We have compared the effect of MIP-1alpha on myeloid and erythroid colony formation of CD34+ cells isolated from bone marrow and cord blood. In the presence of MIP-1alpha, bone marrow granulocyte-macrophage-colony forming cells (GM-CFC) were inhibited over a dose range of 15 ng/ml to 500 ng/ml, and GM-CFC from cord blood CD34+ cells were stimulated over the same dose range. MIP-1alpha suppressed BFU-E colonies in both bone marrow and cord blood. Using thymidine suicide assays, the influence of MIP-1alpha on the cycling status of the cells was assessed. A good correlation between the effect of MIP-1alpha on colony formation and cell cycle progression was observed. These results suggest that there is a differential response to MIP-1alpha when bone marrow and cord blood CD34+ cells are compared. Using flow cytometry and a biotinylated human MIP-1alpha/avidin fluorescein conjugate, the expression of MIP-1alpha receptors on CD34+ cells was assessed. The data indicated that there was little quantitative difference in overall expression of receptors (82.9% versus 93%) from bone marrow or cord blood, respectively. However, when Northern blot analysis was used, mRNA for two different MIP-1alpha receptors CCR1 and CCR5 could be detected in bone marrow, but only CCR1 mRNA was seen in cord blood CD34+ samples. Therefore, the expression of different receptor subtypes on CD34+ cells may be responsible for the difference in MIP-1alpha responsiveness observed.

Randomized comparison of progenitor-cell mobilization using chemotherapy, stem-cell factor, and filgrastim or chemotherapy plus filgrastim alone in patients with ovarian cancer.

PURPOSE: This was the first randomized study to investigate the efficacy of peripheral-blood progenitor cell (PBPC) mobilization using stem-cell factor (SCF) in combination with filgrastim (G-CSF) following chemotherapy compared with filgrastim alone following chemotherapy. PATIENTS AND METHODS: Forty-eight patients with ovarian cancer were treated with cyclophosphamide and randomized to receive filgrastim 5 microg/kg alone or filgrastim 5 microg/kg plus SCF. The dose of SCF was cohort-dependent (5, 10, 15, and 20 microg/kg), with 12 patients in each cohort, nine of whom received SCF plus filgrastim and the remaining three patients who received filgrastim alone. On recovery from the WBC nadir, patients underwent a single apheresis. RESULTS: SCF in combination with filgrastim following chemotherapy enhanced the mobilization of progenitor cells compared with that produced by filgrastim alone following chemotherapy. This enhancement was dose-dependent for colony-forming unit-granulocyte-macrophage (CFU-GM), burst-forming unit-erythrocyte (BFU-E), and CD34+ cells in both the peripheral blood and apheresis product. In the apheresis product, threefold to fivefold increases in median CD34+ and progenitor cell yields were obtained in patients treated with SCF 20 microg/kg plus filgrastim compared with yields obtained in patients treated with filgrastim alone. Peripheral blood values of CFU-GM, BFU-E, and CD34+ cells per milliliter remained above defined threshold levels longer with higher doses of SCF. The higher doses of SCF offer a greater window of opportunity in which to perform the apheresis to achieve high yields. CONCLUSION: SCF (15 or 20 microg/kg) in combination with filgrastim following chemotherapy is an effective way of increasing progenitor cell yields compared with filgrastim alone following chemotherapy.

A randomized phase-II study of BB-10010 (macrophage inflammatory protein- 1alpha) in patients with advanced breast cancer receiving 5-fluorouracil, adriamycin, and cyclophosphamide chemotherapy.

BB-10010 is a variant of the human form of macrophage inflammatory protein-1alpha (MIP-1alpha), which has been shown in mice to block the entry of hematopoietic stem cells into S-phase and to increase their self-renewal capacity during recovery from cytotoxic damage. Its use may constitute a novel approach for protecting the quality of the stem cell population and its capacity to regenerate after periods of cytotoxic treatment. Thirty patients with locally advanced or metastatic breast cancer were entered into the first randomized, parallel group controlled phase II study. This was designed to evaluate the potential myeloprotective effects of a 7-day regimen of BB-10010 administered to patients receiving six cycles of 5-fluorouracil (5-FU), adriamycin, and cyclophosphamide (FAC) chemotherapy. Patients were randomized, 10 receiving 100 microgram/kg BB-10010, 11 receiving 30 microgram/kg BB-10010, and nine control patients receiving no BB-10010. BB-10010 was well-tolerated in all patients with no severe adverse events related to the drug. Episodes of febrile neutropenia complicated only 4% of the treatment cycles and there was no difference in incidence between the treated and nontreated groups. Studies to assess the generation of progenitor cells in long-term bone marrow cultures were performed immediately preceding chemotherapy and at the end of six dosing cycles in 18 patients. Circulating neutrophils, platelets, CD 34(+) cells, and granulocyte/macrophage colony-forming cell (GM-CFC) levels were determined at serial time points in cycles 1, 3, and 6. The results showed similar hemoglobin and platelet kinetics in all three groups. On completion of the six treatment cycles, the average pretreatment neutrophil levels were reduced from 5.3 to 1.7 x 10(9)/L in the control patients and from 4.3 to 1.9 and 4.5 to 2.5 x 10(9)/L in the 30/100 microgram/kg BB-10010 groups, respectively. Relative to their pretreatment values, 50% of the patients receiving BB-10010 completed the treatment with neutrophil values significantly higher than any of the controls (P = .02). Mobilization of GM-CFC was enhanced by BB-10010 with an additional fivefold increase over that generated by chemotherapy alone, giving a maximal 25-fold increase over pretreatment values. Bone marrow progenitor assays before and after this standard regimen of chemotherapy indicated little long-term cumulative impairment to recovery from chemotherapy. Despite the limited cumulative damage to the bone marrow, which may have minimized the protective value of BB-10010 during this regimen of chemotherapy, better recovery of neutrophils in the later treatment cycles with BB-10010 was indicated in a number of patients.