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

The engraftment capacity of bone marrow–derived mesenchymal cells was investigated in 41 patients who had received a sex-mismatched, T-cell–depleted allograft from human leukocyte antigen (HLA)–matched or –mismatched family donors. Polymerase chain reaction (PCR) analysis of the human androgen receptor (HUMARA) or the amelogenin genes was used to detect donor-derived mesenchymal cells. Only 14 marrow samples (34%) from 41 consenting patients generated a marrow stromal layer adequate for PCR analysis. Monocyte-macrophage contamination of marrow stromal layers was reduced below the levels of sensitivity of HUMARA and amelogenin assays (5% and 3%, respectively) by repeated trypsinizations and treatment with the leucyl-leucine (leu-leu) methyl ester. Patients who received allografts from 12 female donors were analyzed by means of the HUMARA assay, and in 5 of 12 cases a partial female origin of stromal cells was demonstrated. Two patients who received allografts from male donors were analyzed by amplifying the amelogenin gene, and in both cases a partial male origin of stromal cells was shown. Fluorescent in situ hybridization analysis using a Y probe confirmed the results of PCR analysis and demonstrated in 2 cases the existence of a mixed chimerism at the stromal cell level. There was no statistical difference detected between the dose of fibroblast progenitors (colony-forming unit–F [CFU-F]) infused to patients with donor- or host-derived stromal cells (1.18 ± 0.13 × 104/kg vs 1.19 ± 0.19 × 104/kg; P ≥ .97). In conclusion, marrow stromal progenitors reinfused in patients receiving a T-cell–depleted allograft have a limited capacity of reconstituting marrow mesenchymal cells.