Summary: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. /l white blood cells and 50 ؋ 10 9 /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.

Summary:Keywords: hematopoietic progenitors; microenvironmental progenitors; acute myelogenous leukemia; chemotherapy; LTC-IC; hematopoietic engraftment Since reduced marrow cellularity and prolonged pancytopenia following autologous bone marrow transplantation (ABMT) have been frequently observed in patients with acute myelogenous leukemia (AML)The structural integrity of the hematopoietic system is included in the AML10 GIMEMA/EORTC trial, the maintained by a relatively small population of selfquestion was raised to what extent hematopoietic and renewing stem cells which can differentiate to produce promicroenvironmental progenitor cells were involved in genitors committed to terminal maturation. 1 The developthese patients. Marrow hematopoietic progenitors were ment of hematopoietic cells in vivo occurs in intimate investigated by a short-term methylcellulose assay association with a heterogeneous population of mesenchyquantitating multipotent CFU-Mix, erythroid BFU-E mal, connective tissue type cells and their associated and granulocyte-macrophage CFU-GM, as well as a biosynthetic products, which constitute the stromal tissue long-term assay quantitating long-term cultureof the bone marrow. Stromal cells of the hematopoietic initiating cells (LTC-IC). The marrow microenviron-microenvironment include fibroblasts, endothelial cells, ment was studied by evaluating the incidence of fibroadipocytes, and macrophages. 2 Based on a number of studblastoid progenitors (CFU-F) and the capacity of stroies, 3 the existence of self-renewing stromal stem cells with mal layers to support allogeneic hematopoietic multilineage differentiation capacity and capable of generprogenitors. As compared to normal controls (n = 57), ating progenitors with restricted development potential, AML patients (n = 26) showed a statistically significant including fibroblast, osteoblast and chondrocyte progenireduction of the mean (± s.e.m.) number of CFU-Mix tors, has been hypothesized. 4-6(5.3 ± 0.6 vs 0.8 ± 0.2, P р 0.0001), BFU-E (68 ± 5 vs Standard-and high-dose therapies currently used for the 20 ± 4, P р 0.0001), CFU-GM (198 ± 11 vs 144 ± 15, P treatment of hematological and nonhematological malig-р 0.008), and LTC-IC (302 ± 46 vs 50 ± 8, P р 0.001).nancies induce transient or permanent damage of hematoThe mean (± s.e.m.) incidence of marrow CFU-F was poietic and stromal progenitor cell compartments. 7,8not significantly reduced as compared to normal conDespite such chemotherapy-induced defective progenitor trols (48 ± 6 vs 52 ± 7, P р 0.73). Seventeen AML strocell growth, the reinfusion of autologous marrow can reconmal layers were tested for their capacity to support the stitute the hematopoietic system, even in acute myelogengrowth of allogeneic hematopoietic progenitors. Seven ous leukemia (AML) patients treated with remission inducsamples failed to support any progenitor cell growth, tion regimens exerting a significant marrow toxicity. 9,10seven had a significantly lower supportive activity as Recently, therapeutic trials have been...

Summary: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 ؋ 10 6 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 ؋ 10 8 vs 2.53 ؎ 0.4 ؋ 10 8 , P р 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 ؋ 10 9 vs 32 ؎ 5.4 ؋ 10 9 , P р 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 р 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 ؋ 10 6 vs 14 ؎ 2 ؋ 10 6 , P р 0.03). Interestingly, CY+G-CSF-mobilized CD34 ؉ cells had a significantly higher plating efficiency than G-CSF-mobilized CD34 ϩ cells (25.5 ؎ 2.9% vs 10.8 ؎ 1.9%, P р 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 ؋ 10 6 vs 3.3 ؎ 0.3 ؋ 10 6 , P р 0.05). In conclusion, our data provide evidence that CY؉G-CSF and G-CSF induce the mobilization of CD34 ؉ cells with

SummaryFetal blood collected immediately after delivery has been was the aim of this study to evaluate whether cryopreshown to contain hematopoietic progenitor cells at similar servation procedures might heavily impair the clonoor higher frequency than those in bone marrow (BM).1 genic capacity, the feasibility of CD34 ؉ selection and the Therefore, umbilical cord blood (UCB), which is normally ex vivo expansion potential of UCB progenitor cells.discarded, has been evaluated as a source of UCB samples were collected and cryopreserved as stem/progenitor cells 2-4 that can easily be collected at delivunseparated (n ‫؍‬ 21) or mononuclear (MNC) cells (n ery without any danger or inconvenience to the donor. 5,6‫؍‬ 15) within 12 h from delivery, and evaluated for Recently, UCB has been used as a source of hematopoietic viability, immunophenotype, cell and progenitor numstem cells for clinical transplantation, and is proving to be bers after a minimum stay in liquid nitrogen of 6 an acceptable alternative to BM. 7-9 Several studies have months (range 6-14 months). Viability was always demonstrated that UCB contains similar or higher pro-Ͼ97% and no statistically significant difference was portions of primitive hematopoietic progenitor cells as detected by flow cytometric analysis. Clonogenic recovcompared to adult BM 2,4 and therefore the lower number ery from unseparated cells was 80-87% for HPP-CFC, of nucleated cells, present in a single collection, might be CFU-GEMM, BFU-E and CFU-GM, and from MNC compensated by a significantly higher proportion of cells ranged from 82 to 91% for LTC-IC, CFU-GEMM, primitive cells. More recently, highly purified BFU-E and CFU-GM. CD34؉ selection (n ‫؍‬ 8) was per-CD34 capacity has made UCB-derived progenitor cells ideal cansignificant difference was detected in CFC fold-expandidates for experimental programs involving gene transfer sion for fresh or cryopreserved MNC cells and for and ex vivo stem cell expansion. Since several programs CD34 ؉ cells, either selected and cultured from fresh or throughout Europe and the USA are currently evaluating cryopreserved MNC cells. In conclusion we can state the feasibility of large-scale UCB banking for unrelated that UCB is a potential source of primitive progenitor transplants 5,12,13 cytokine-mediated ex vivo expansion of cells that can be cryopreserved unmanipulated or after UCB hematopoietic progenitor cells might increase the physical separation without major losses in clonogenic number of progenitor cells to be transplanted and facilitate capacity and immunophenotypic composition. Moreengraftment in adult patients. Therefore, it was the aim of our study to investigate whether cryopreservation can affect the clonogenic capacity, the immunophenotype, the feasi- potential of UCB progenitor cells.

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 SCF low and 8.1 ± 1.5% as SCF high . 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 р 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 р 0.018) and mafosfamide-treated samples (62 ± 5.6% vs 44 ± 6.1%, P р 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.