Lina Mangoni

CD34 + cells mobilized by cyclophosphamide and granulocyte colony-stimulating factor (G-CSF) are functionally different from CD34 + cells mobilized by G-CSF

Mobilized peripheral blood progenitor cells (PBPC) are increasingly used as an alternative to bone marrow for autografting procedures. Currently, cyclophosphamide (CY) followed by granulocyte colony-stimulating factor (G-CSF) or G-CSF alone are the most commonly used PBPC mobilization schedules. In an attempt to investigate whether the use of these two mobilization regimens could result in the collection of functionally different CD34+ cells, we analyzed nucleated cells (NC), CD34+ cells, committed progenitor cells and long-term culture initiating-cells (LTC-IC) in 52 leukaphereses from 26 patients with lymphoid malignancies, mobilized either by CY+G-CSF (n = 16) or G-CSF alone (n = 10). Thirty-four aphereses from the CY+G-CSF group and 18 aphereses from the G-CSF group were investigated. According to the study design, leukaphereses were carried out until an average number of 7 × 106 CD34+ cells/kg body weight were collected. The mean (± s.e.m.) numbers of CD34+ cells mobilized per apheresis by CY+G-CSF and G-CSF were not significantly different (2.76 ± 0.6 × 108 vs 2.53 ± 0.4 × 108, P ⩽ 0.7). This resulted from a mean number of NC that was significantly lower in the CY+G-CSF products than in the G-CSF products (12.4 ± 1.7 × 109 vs 32 ± 5.4 × 109, P ⩽ 0.0001) and a mean incidence of CD34+ cells that was significantly higher in the CY+G-CSF products than in the G-CSF products (2.9 ± 0.6% vs 0.9 ± 0.2%, P ⩽ 0.0018). The mean (± s.e.m.) number of CFU-GM collected per apheresis was significantly higher in the CY+G-CSF group than in the G-CSF group (37 ± 7 × 106 vs 14 ± 2 × 106, P ⩽ 0.03). Interestingly, CY+G-CSF-mobilized CD34+ cells had a significantly higher plating efficiency than G-CSF-mobilized CD34+ cells (25.5 ± 2.9% vs 10.8 ± 1.9%, P ⩽ 0.0006). In addition, the mean number of LTC-IC was significantly higher in the CY+G-CSF products than in the G-CSF products (6.3 ± 1 × 106 vs 3.3 ± 0.3 × 106, P ⩽ 0.05). In conclusion, our data provide evidence that CY+G-CSF and G-CSF induce the mobilization of CD34+ cells with different clonogenic potential. As mobilized PBPC containing large numbers of progenitors lead to safer transplantation, this issue may have implications for planning mobilization strategies.

Clonogenic capacity and ex vivo expansion potential of umbilical cord blood progenitor cells are not impaired by cryopreservation

Umbilical cord blood (UCB) progenitor cells have been demonstrated to possess significant advantages over bone marrow (BM), in terms of proliferative capacity and immunologic reactivity. Therefore, UCB has been recently considered an attractive potential alternative to BM as a source of hematopoietic progenitor cells for clinical applications. Since several programs throughout the world are currently evaluating the feasibility of large-scale UCB banking for unrelated transplants, it was the aim of this study to evaluate whether cryopreservation procedures might heavily impair the clonogenic capacity, the feasibility of CD34+ selection and the ex vivo expansion potential of UCB progenitor cells. UCB samples were collected and cryopreserved as unseparated (n = 21) or mononuclear (MNC) cells (n = 15) within 12 h from delivery, and evaluated for viability, immunophenotype, cell and progenitor numbers after a minimum stay in liquid nitrogen of 6 months (range 6–14 months). Viability was always >97% and no statistically significant difference was detected by flow cytometric analysis. Clonogenic recovery from unseparated cells was 80–87% for HPP-CFC, CFU-GEMM, BFU-E and CFU-GM, and from MNC cells ranged from 82 to 91% for LTC-IC, CFU-GEMM, BFU-E and CFU-GM. CD34+ selection (n = 8) was performed on fresh and cryopreserved MNC cells using the MiniMACS immunomagnetic separation device, showing no difference in yield (68 ± 7% vs 57 ± 4%, P ⩽ 0.4) or in purity (89 ± 2% vs 81 ± 6%, ⩽ 0.4), for fresh in comparison to cryopreserved MNC cells. After 14 days of liquid culture in the presence of different combinations of SCF, IL-3, IL-6 and G-CSF no statistically significant difference was detected in CFC fold-expansion for fresh or cryopreserved MNC cells and for CD34+ cells, either selected and cultured from fresh or cryopreserved MNC cells. In conclusion we can state that UCB is a potential source of primitive progenitor cells that can be cryopreserved unmanipulated or after physical separation without major losses in clonogenic capacity and immunophenotypic composition. Moreover, CD34+ selection from cryopreserved MNC cells is feasible and ex vivo expansion is not impaired. These results have important implications in the large scale UCB banking, in view of the potential applications of ex vivo expanded hematopoietic progenitor cells for the engraftment of adult patients.

Primitive hematopoietic progenitors within mobilized blood are spared by uncontrolled rate freezing.

Uncontrolled-rate freezing techniques represent an attractive alternative to controlled-rate cryopreservation procedures which are time-consuming and require high-level technical expertise. In this study, we report our experience using uncontrolled-rate cryopreservation and mechanical freezer storage at −140°C. Twenty-eight PBPC samples (10 cryovials, 18 freezing bags) from 23 patients were cryopreserved in a cryoprotectant solution composed of phosphate-buffered saline (80%, v/v) supplemented with human serum albumin (10%, v/v) and dimethylsulfoxide (10%, v/v). The cryopreservation procedure required on average 1.5 h. The mean (± s.e.m.) storage time of cryovials and bags was 344 ± 40 and 299 + 57 days, respectively. Although cell thawing was associated with a statistically significant reduction of the absolute number of nucleated cells (vials: 0.3 × 109 vs 0.2 × 109, P ⩽ 0.02; bags: 14 × 109 vs 11 × 109, P ⩽ 0.0003), the growth of committed progenitors was substantially unaffected by the freezing–thawing procedure, with mean recoveries of CFU-Mix, BFU-E, and CFU-GM ranging from 60 ± 29% to 134 ± 15%. Mean recoveries of LTC-IC from cryovials and bags were 262 ± 101% and 155 ± 27% (P ⩽ 0.2), respectively. In 14 out of 23 patients who underwent high-dose chemotherapy and PBPC reinfusion, the pre- and post-freezing absolute numbers of hematopoietic progenitors cryopreserved in bags were compared. A significant reduction was detected for CFU-Mix (11 vs 7.4 × 105), but no significant loss of BFU-E (180 vs 150 × 105), CFU-GM (400 vs 290 × 105) and LTC-IC (15 vs 16 × 105) could be demonstrated. When these patients were reinfused with uncontrolled-rate cryopreserved PBPC, the mean number of days to reach 1 × 109/l white blood cells and 50 × 109/l platelets were 9 and 13, respectively. In conclusion, the procedure described here is characterized by short execution time, allows a substantial recovery of primitive and committed progenitors and is associated with prompt hematopoietic recovery following myeloablative therapy even after long-term storage.

Biologic and phenotypic analysis of early hematopoietic progenitor cells in umbilical cord blood

Umbilical cord blood (UCB) is an attractive potential alternative to bone marrow (BM) as a source of hematopoietic progenitor cells since the number of progenitors in UCB is similar or even greater than that in normal BM. It was the aim of the present study to analyze the degree of immaturity of UCB progenitor cells. UCB mononuclear (MNC) and/or CD34+ cells were tested for surface antigen phenotype, expression of cytokines receptor, effect of stem cell factor (SCF) on colony growth, resistance to mafosfamide and replating potential. We have found that 34.9 ± 3.4% and 77.9 ± 2.6% of UCB CD34+ cells did not express CD38 and CD45RA antigens, respectively, suggesting that UCB contains a high proportion of immature progenitor cells. By means of three-color analysis, the receptor for SCF was detected on the majority of the CD34+HLA-DR+ subpopulation; in fact, 81.8% ± 4.3% of CD34+HLA-DR+ cells were defined as SCFlow and 8.1 ± 1.5% as SCFhigh. Colony growth of MNC and CD34+ cells was enhanced by the addition of SCF to methylcellulose mixture, resulting in a statistically significant increase in CFU-GM and CFU-GEMM but not in BFU-E numbers. UCB progenitor cells showed a higher resistance to mafosfamide treatment, in comparison to BM; the addition of SCF to the culture medium resulted in a statistically significant increase in mafosfamide concentration required to inhibit 95% of colony growth (P ⩽ 0.05). Moreover, as shown by single colony transfer assays, the presence of SCF in primary cultures promoted a significantly higher replating potential for both untreated (42 ± 3.3% vs 21 ± 4.6%, P ⩽ 0.018) and mafosfamide-treated samples (62 ± 5.6% vs 44 ± 6.1%, P ⩽ 0.018). In conclusion, UCB is a source of progenitor cells with immature characteristics in terms of surface antigen expression, distribution of SCF receptor, resistance to mafosfamide and replating potential. Therefore, UCB progenitor cells represent an ideal candidate population for experimental programs involving gene transfer and ex vivo stem cell expansion.

Effect of the protein tyrosine kinase inhibitor genistein on normal and leukaemic haemopoietic progenitor cells

Receptor and nonreceptor protein tyrosine kinases (PTKs) play a key role in the control of normal and neoplastic cell growth. The availability of PTK inhibitors prompted us to evaluate the effects of genistein, a natural inhibitor of PTKs, on in vitro colony formation by normal multilineage colony‐forming units (CFU‐Mix), erythroid bursts (BFU‐E), granulocyte‐macrophage colony‐forming units (CFU‐GM), long‐term culture‐initiating cells (LTC‐IC) and acute myelogenous leukaemia colony‐forming units (CFU‐AML). Continuous exposure of normal marrow and blood mononuclear non‐adherent cells, blood CD34+CD45RA− cells, and leukaemic blasts to increasing doses of genistein (1–100 μM) resulted in a statistically significant (P ≤ 0.05) dose‐dependent suppression of CFU‐Mix, BFU‐E, CFU‐GM and CFU‐AML growth. Regression analysis showed that growth inhibition was linearly related to genistein concentration. Genistein dose causing 50% inhibition (ID50) of CFU‐AML was significantly lower compared to CFU‐GM ID50 for marrow (19 v 32 μMP  ≤0.017), unseparated blood (19 v 44 μMP ≤ 0.028) or CD34+CD45RA− blood (19 v 36, P ≤ 0.04). Preincubation of leukaemic blasts with genistein (200 μM) for 1–2 h confirmed that CFU‐AML were significantly more sensitive than normal marrow and blood CFU‐GM to genistein. Preincubation conditions which maximally suppressed leukaemic and normal colony growth spared a substantial percentage of marrow (29 ± 4%) and blood (40 ± 3%) LTC‐IC. In conclusion, our data demonstrate that: (a) genistein strongly inhibits the growth of normal and leukaemic haemopoietic progenitors; (b) growth inhibition is dose‐ and time‐dependent; (c) leukaemic progenitors are more sensitive than normal progenitors to genistein‐induced growth inhibition; (d) genistein exerts a direct toxic effect on haemopoietic cells while sparing a substantial proportion of LTC‐IC. The potent CFU‐AML growth inhibition associated with the relative resistance of normal LTC‐IC strongly supports the use of genistein for marrow purging.

Long‐term outcome of adults with acute myelogenous leukaemia: results of a prospective, randomized study of chemotherapy with a minimal follow‐up of 7 years

Summary. In a prospective study running between 1981 and 1983, a group of 156 adult (under 60 years of age) patients with de‐novo acute myelogenous leukaemia were randomly assigned to receive a daunorubicin, cytosine arabinoside and thioguanine combination or a regimen containing lower dosages of these drugs but also containing etoposide and vindesine. Patients who entered complete remission received maintenance therapy for 2 years. The survival and remission duration curves of the two groups were exactly superimposable and for this long‐term analysis all patients have been considered together. The follow‐up times range between 84 and 104 months. Actual survival at 7 years is 15% (95% confidence intervals 9–20%), with a stable curve thereafter. Actual probability of continuous complete remission at 7 years is 22% (95% C.I. 13–31%), with a stable curve beyond that point. These findings, similar to those of the few other studies of chemotherapy with comparable follow‐up times, suggest that only a small fraction of adult patients become long‐term survivors, irrespective of the precise type or amount of antineoplastic agents administered.

Techniques for detection of minimal residual disease.

Analysis of leukemia-specific and leukemia-associated markers following standard or high-dose treatments is crucial in order to evaluate the efficacy of therapeutic strategies. During the last decade, several techniques have been proposed and used for detecting minimal residual disease (MRD). Each approach is characterized by advantages and limitations, mainly related to its sensitivity and specificity. The general limitations of such tests originates from the size of the sample which can be analysed and the heterogeneous distribution of leukemia after treatment. Clinically useful methods for detecting residual leukemia require not only sensitivity but also speed and reproducibility. The rate of false negative tests is low with polymerase chain reaction as well as flow cytometric analysis. Usually, patients without persistent cells carrying leukemia-associated markers have a lower risk of relapse. However, the detection of a persistent marker at one time point in complete remission cannot be considered a reliable indicator of MRD, whereas increase of positive signals or reappearance of leukemic markers usually precedes relapse. It is likely that one single approach will not allow the monitoring of the majority of patients and that a combination of techniques will be needed. Definitive results will be obtained only through prospective studies in patients receiving standardised therapy. Studies in which therapeutic strategies are designed according to the results provided by techniques for detecting MRD will be necessary to assess the relevance of their contribution to the treatment of leukemia.

Assessment of the efficacy of a last-generation polyvalent immunoglobulin in the treatment of idiopathic thrombocytopenic purpura

High-dose intravenous immunogammaglobulin (h.d.IgG) has been proposed as a treatment of idiopathic thrombocytopenic purpura (ITP), but the clinical effect is usually short and adverse reactions have been reported in clinical studies using different immunoglobulin (Ig) preparations. In this study, the efficacy of a last-generation polyvalent immunoglobulin in the treatment of ITP in adults and the incidences of adverse reactions of this therapy were evaluated. The reported data were based on various clinical and laboratory parameters evaluated before, during and after therapy, with a follow-up of 6 months. The data showed administration of 400 mg/kg d of intravenous polyvalent intact IgG for 5 days significantly increased the platelet count in all 15 patients, the maximum level occurring on Day 10 and being maintained in some patients for 6 months. Its very rapid onset of action suggests it may be useful for correcting life-threatening thrombocytopenia where bleeding complicates the clinical course, and for severe ITP in seriously immunosuppressed or infected patients in whom corticosteroids or immunosuppressive agents cannot be safely administered. The treatment was also well tolerated. In conclusion, polyvalent Ig may be useful in ITP steroid-refractory patients; further studies are required to evaluate clinical-laboratory parameters related to the long-term response of patients.

Biology and clinical applications of long-term bone marrow cultures.

A number of clonogenic assays for short-term bone marrow culture is now available for the quantitative analysis of the various hematopoietic progenitor cell classes. The short-term assays are not suitable to analyse either stem cell selfrenewal or interactions of hematopoietic progenitors with stromal cells, especially those requiring direct cell-to-cell or cell-to-matrix contact. The technique of longterm bone marrow culture (LTBMC) allows a sustained production of myeloid cells when marrow is placed in liquid culture at relatively high cell concentration, with appropriate supplements, temperature and feeding conditions. A peculiar feature of LTBMC is that the stromal cells promote selfrenewal as well as differentiation of the stem cells, without the need to add exogenous growth factors. The LTBMC system offers an approach able to investigate not only the proliferative and differentiative events but also sustained cell production and selfrenewal of any clonogenic cell types. In the last years, the technique of LTBMC has been increasingly used by several groups to investigate hematopoietic regulation, stromal cell function and the interactions among stromal and hematopoietic cells. In the present report, the biology of LTBMC and their possible clinical applications will be reviewed.

In vitro growth of mobilized peripheral blood progenitor cells is significantly enhanced by stem cell factor.

The existence of primitive hematopoietic progenitors in mobilized peripheral blood is suggested by clinical, phenotypic and in vitro cell culture evidences. In order to quantify primitive progenitors, 32 leukaphereses from 15 patients with lymphoid malignancies were investigated for the growth of multilineage colony‐forming units (CFU‐Mix), erythroid burst‐forming units (BFU‐E) and granulocyte‐macrophage colony‐forming units (CFU‐GM) in the absence or presence of recombinant stem cell factor (SCF), a cytokine which selectively controls stem cell self‐renewal, proliferation and differentiation. Primitive progenitors were also quantitated by means of a long‐term assay which allows the growth of cells capable of initiating and sustaining hematopoiesis in long‐term culture (LTC‐IC). Addition of SCF (50 ng/ml) to methylcellulose cultures stimulated with maximal concentrations of G‐CSF, GM‐CSF, interleukin 3 and erythropoietin significantly increased the growth (mean ± SE) of CFU‐Mix (7.7 ± 1.7 versus 2.4 ± 0.6, p ≤ 0.0001), BFU‐E (47 ± 10 versus 32 ± 6, p ≤ 0.002) and CFU‐GM (173 ± 31 versus 112 ± 20, p ≤ 0.0001). Mean (± SE) percentages of SCF‐dependent CFU‐Mix, BFU‐E and CFU‐GM were 60 ± 5%, 19 ± 5%, and 33 ± 4%, respectively. Mean (± SE) LTC‐IC growth per 2 × 106 nucleated cells was 221 ± 53 (range, 2 to 704). Linear regression analysis demonstrated a statistically significant correlation (r =.87; p ≤ 0.0001) between LTC‐IC and SCF‐dependent progenitors. In conclusion, our data suggest that: A) the optimal quantification of mobilized progenitors requires supplementation of methylcellulose cultures with SCF, and B) in vitro detection of SCF‐dependent progenitors might represent a reliable and technically simple method to assess the primitive progenitor cell content of blood cell autografts. Such in vitro evaluation of immature hematopoietic progenitors might be clinically relevant for predicting the reconstituting potential of autografts.