D. Garau

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.

Reverse transcription polymerase chain reaction is a reliable assay for detecting leukemic colonies generated by chronic myelogenous leukemia cells

Single-colony karyotyping (SCK) and reverse-transcription polymerase chain reaction (RT-PCR) are two increasingly used techniques for the quantification of leukemic colonies generated by chronic myelogenous leukemia (CML) cell fractions purged or selected in vitro. Recently, the existence of Philadelphia (Ph) chromosome positive progenitors with a silent BCR-ABL gene has been reported, thus raising concerns on the use of RT-PCR for detecting BCR-ABL positive progenitors. In order to investigate this issue further, colonies (n = 204) generated by mononuclear (MNC) or CD34+ CML cells were individually harvested, divided into two aliquots and analyzed both at the cytogenetic level to detect the Ph chromosome, and the molecular level to detect BCR-ABL transcripts. The mean (± s.d.) percentages of colonies analyzable by either SCK or RT-PCR were 74 ± 16% and 86 ± 16%, respectively. A significant percentage of colonies (67 ± 19%) could be successfully analyzed by both SCK and RT-PCR. Although the majority of these colonies (97 ± 5%) were Ph-positive and BCR-ABL-positive, a negligible percentage (4%) of progenitors were Ph-positive but BCR-ABL-negative. In order to test the influence of colony size on the outcome of molecular analysis, the efficiency of our RT-PCR assay in detecting BCR-ABL transcripts was investigated by means of experiments in which the number of cells used to start RNA extraction was serially reduced. These experiments showed that at least 150 cells were necessary to achieve a reproducible amplification of BCR-ABL transcripts. By correlating the size of harvested colonies with the outcome of molecular analysis, it was evident that BCR-ABL-negative but Ph-positive colonies represented false negative results occurring when a number of leukemic cells below the detection limit of our RT-PCR assay was analyzed. In conclusion, our data demonstrate that individual CML colonies grown in semisolid culture assays can be indifferently analyzed by SCK or RT-PCR, and support an extensive use of a carefully standardized RT-PCR assay to estimate the leukemic burden within samples which have been purged and selected in vitro.

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.

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.

Fractionation of chronic myelogenous leukemia marrow cells by stroma adherence: Implications for marrow purging

Chronic myelogenous leukemia (CML) progenitor cells have been shown to be defective in their ability to adhere to marrow stroma. It was the aim of the present study to investigate at the cytogenetic level marrow-derived CML clonogenic cells fractionated on the basis of their ability to adhere to preformed, allogeneic, normal marrow-derived stromal layers. Mononuclear marrow cells from CML patients (n = 15) were incubated with mafosfamide (100 micrograms/ml) or control medium, seeded onto marrow stromal layers and allowed to adhere (3 hrs, 37 degrees C). Following a short-term liquid culture, the different cell fractions were harvested and incorporated in methylcellulose cultures. CFU-GM grown from these cultures were analyzed by single colony karyotyping. On direct cytogenetic analysis, the overall mean (+/- SD) percentage of Ph-negative metaphases was 7 +/- 20%. Following stroma adherence and shortterm suspension culture, the mean (+/- SD) percentages of Ph-negative clones were as follows: 33 +/- 25% for adherent CFU-GM, 59 +/- 40% for adherent, mafosfamide-treated CFU-GM, 12 +/- 16% for non-adherent CFU-GM, and 32 +/- 26% for non-adherent-mafosfamide-treated CFU-GM. If only the patients showing a percentage of Ph-negative clones > or = 20% were included in this analysis, the mean (+/- SD) percentages of Ph-negative clones were 47 +/- 19% for adherent CFU-GM, and 81 +/- 21% for adherent-Mafosfamide-treated CFU-GM. In contrast, the majority of pH-positive CFU-GM were detected within the stroma non-adherent cell fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Shc Overexpression Induces Selective Hypersensitivity to GM-CSF and Prevents Apoptosis of the GM-CSF-Dependent Acute Myelogenous Leukemia Cell Line GF-D8

Hematopoietic cell self-renewal, proliferation, differentiation, and survival are regulated by a complex network of integrated processes under the control of extra- and intra-cellular mechanisms. Shc is an adaptor molecule implicated in the regulation of cell proliferation. To assess the role Shc may play in regulating hematopoietic cells, we engineered the overexpression of Shc in the recently described GM-CSF-dependent GF-D8 cell line by retroviral gene transfer and analyzed subsequent effects on cell behavior. Early passaged Shc- or mocktransfected GF-D8 clones, cultured in RPMI1640 with FBS (10%) and GM-CSF (20 ng/ml), were used. Western blot analysis confirmed that the transfected clone (GFD8/Shc) had a significantly higher expression of Shc as compared to the parental clone (GF-D8), or clones retrovirally transduced with the LXSN vector only (GFD8/SN). Analysis of nuclear DNA fragmentation by DNA gel electrophoresis revealed that GF-D8/SN cells underwent apoptosis 24 h following GM-CSF deprivation, whereas GF-D8/Shc cells failed to show any evidence of apoptosis up to six days after GM-CSF deprivation. Apoptosis was associated with a progressive decrease of Bd-2 and increase of CD95 expression. To evaluate the clonogenic response of GF-D8/SN and GF-D8/Shc to growth factors, cells were GM-CSF-starved for 24–48 h and then cultured in methylcellulose with increasing concentrations (0.001–50 ng/ml) of different growth factors, including GM-CSF, G-CSF, and MGDF. Cell proliferation was analyzed by assaying colonies (aggregates of ≥ 40 cells). As compared to GF-D8/Shc cells, GF-D8/SN cells generated significantly lower numbers of colonies upon stimulation with GM-CSF. Both Shcor mock-transfected GF-D8 cells failed to give rise to clonal aggregates in response to G-CSF and MGDF. In conclusion, our data demonstrate that Shc overexpression increases GM-CSF sensitivity and prevents apoptosis of GF-D8 cells. These results suggest that: 1. Shc is an important regulator of cell survival and proliferation, 2. our model system might be useful for investigating leukemic hematopoiesis as well as the controlled amplification of genetically modified hematopoietic cells.