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Background Aims: The aim of this study was to compare prospectively the vasculogenic capacity of two cell sources, monocytes and CD133+ cells., Methods: Cells were obtained from healthy donors by adherence or magnetic selection. Animals studies were performed in a model of hind limb ischemia and different groups were established according to type and number of cells infused. Revascularization was measured by sequential blood flow analysis using a laser Doppler device and by assessing capillary density in the ischemic muscles. In order to locate the infused cells, immunofluorescence and immunocytochemistry techniques were performed and analyzed by light and confocal microscopy., Results: During the study period there was a significant improvement in both limb perfusion and capillary density in mice treated with either human monocytes or CD133+ cells (P<0.05) compared with non-treated mice. No cells were detected as incorporated into the vessels when 1 x 10(5) cells were used but with higher doses (1 x 10(6)) a few human cells were observed integrated into the vessels in both groups of treated mice. Supernatants of both cell types showed vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and platelet-derived growth factor- AB (PDGF-AB) expression., Conclusions: Treatment with human monocytes or CD133+ cells improves blood perfusion and capillary density in a murine model and both cell types seem to stimulate vasculogenesis in a fairly similar way.

Objective: Optimization of the mesenchymal stem cells (MSC) isolation and expansion method., Materials and Methods: Mononuclear cells (MNC) from bone marrow aspirates were obtained by both density gradient centrifugation (standard method) and gravity sedimentation. Cells were cultured in standard conditions (10% fetal calf serum and normal oxygen tension [21% O(2)]) and expansion results compared to those obtained with the same culture conditions to which platelet lysate (PL) preparations were added; in addition, the 21% O(2) concentration was compared to a lower (5%) concentration (hypoxia) until the fourth cell passage. Time of expansion, number of cells obtained, morphology, cell surface markers, and differentiation potential were evaluated., Results: MSC obtained by any of the different culture conditions expressed comparable immunophenotype and were able to differentiate into osteoblasts, adipocytes, and chondrocytes. When the number of MSC obtained at fourth passage was analyzed, the highest cell numbers were obtained with gravity sedimentation isolation and PL-supplemented culture and the expansion time was the shortest when cells were cultured under hypoxic conditions., Conclusion: MSC isolation by MNC gravity sedimentation together with culture medium supplementation with 5% of PL in a hypoxic atmosphere (5% O(2)) significantly improved MSC yield and reduced expansion time compared to the standard accepted protocols.

We explored the ability of the proteasome inhibitor bortezomib, which prevents nuclear factor kappaB (NF-kappaB) activation, to block T-cell activation, proliferation, and survival within alloreactive compared with resting T cells. For this purpose, T cells were stimulated with PHA, alphaCD3/alphaCD28, or allogeneic dendritic cells or through mixed lymphocyte cultures. NF-kappaB expression increased in activated T lymphocytes compared with resting T cells. Of interest, the higher the NF-kappaB expression, the more intense the proliferative blockade induced by bortezomib. Moreover, after mixed lymphocyte reaction (MLR) cultures, alloreactive T cells were 2 logs more sensitive to bortezomib-induced apoptosis than the resting T-cell counterpart. This effect was due to a selective induction of apoptosis among activated T cells that was related to caspase activation and cleavage of the antiapoptotic bcl-2 protein and was partially abolished by the addition of the pancaspase inhibitor Z-VAD-FMK. In addition, after secondary MLR, the number of activated T cells was significantly reduced among T lymphocytes previously cultured with bortezomib when cells from the same donor were used as stimulating cells. By contrast, when third-party donor cells were used as stimulating cells, no significant differences were observed between T lymphocytes previously exposed or not to the drug, indicating a highly specific depletion of T lymphocytes alloreactive against primary donor antigens. The addition of bortezomib decreased not only the proliferation and viability of activated T lymphocytes but also the levels of IFNgamma and IL-2, which were significantly decreased among activated T cells cultured with bortezomib at doses ranging from 10 to 100 nM. In conclusion, at concentrations reached in the clinical setting, bortezomib induces selective apoptosis and decreases Th1 response among alloreactive T lymphocytes while it barely affects unstimulated T cells. These results establish the basis for the clinical use of bortezomib in the management of graft-versus-host disease (GVHD).

Objective: To determine the feasibility and safety of skeletal myoblast transplantation in patients with chronic myocardial infarction undergoing coronary artery bypass grafting., Methods: Twelve patients with a previous myocardial infarction and ischemic coronary artery disease underwent treatment with coronary artery bypass grafting surgery and intramyocardial injection of autologous skeletal myoblasts cultured with autologous serum. Global and regional cardiac function was assessed by echocardiogram. Fluorine 18 fluorodeoxyglucose and nitrogen 13-ammonia positron emission tomography studies were used to determine cardiac viability and perfusion. A group of historical control patients (n = 14) treated with coronary artery bypass grafting surgery without myoblast transplantation was analyzed., Results: The left ventricular ejection fraction improved from 35.5% +/- 2.3% (mean +/- SEM) before surgery to 55.1% +/- 8.2% at 12 months (P < .01) in the myoblast group and from 33.6% +/- 9.3% to 38.6% +/- 11% in the control group. Regional contractility also improved in the myoblast group, particularly in cardiac segments treated with skeletal myoblasts (wall motion score index: 3.02 +/- 0.17 at baseline vs 1.36 +/- 0.14 at 12 months; P < .0001). Quantitative fluorine 18-fluorodeoxyglucose and nitrogen 13-ammonia positron emission tomography showed an increase in viability and perfusion 12 months after surgery both globally and in segments treated with myoblasts (P = .012 and P = .004). Skeletal myoblast implantation was not associated with adverse events or an increased incidence of cardiac arrhythmias., Conclusions: In patients with previous myocardial infarction, treatment with skeletal myoblasts in conjunction with coronary artery bypass is safe and feasible and is associated with an increased global and regional left ventricular function, improvement in viability, and perfusion of cardiac tissue and no significant incidence of arrhythmias.

Background and Objectives: There is wide interindividual variation in progenitor cell mobilization. The present study was aimed to analyze steady state hematopoiesis in healthy donors and its influence on hematopoietic progenitor cell (HPC) mobilization., Design and Methods: Bone marrow (BM) was aspirated from 72 healthy donors prior to administration of recombinant human granulocyte colony-stimulating factor (G-CSF). Analyses of CD34+ cells and semisolid cultures as well as long-term cultures were performed from BM or leukapheresis products., Results: Male donors showed a higher number of BFU-E (p=0.007) and committed progenitors (p=0.05), a better stromal layer (p=0.02), and higher long-term bone marrow culture (LT-BMC) counts (p<0.05) when compared to those in female donors. When correlating the culture pattern of the BM with the data from the leukapheresis products, we observed that the number of the immature progenitors in BM correlated significantly with both the number of CD34 + cells and CFU-GM in the first leukapheresis. Univariate analysis revealed that the following variables had a beneficial impact on the number of CD34+ cells: male sex, body weight >73 Kg, G-CSF schedule and results of LT-BMC, although in the multivariate analysis only the number of CFU-GM obtained after LT-BMC showed a significant influence (p<0.001)., Interpretation and Conclusions: These results confirm the interindividual variation in HPC mobilization among healthy subjects, with LT-BMC counts being the most reliable predictor, expressing the behavior of the immature progenitors and their relationship with the microenvironment.

Background and Objectives: It has recently been demonstrated that autologous bone-marrow transplantation (ABMT) is feasible in heavily pretreated patients who do not mobilize peripheral blood progenitor cells (PBPC), suggesting that bone marrow (BM) progenitor-cells are not as sensitive to chemotherapy as are PBPC. However, information regarding the impact of previous chemotherapy on the performance of BM grafts is scanty., Design and Methods: We have retrospectively analyzed 40 consecutive lymphoma patients treated with the BEAM regimen and ABMT at our institution. The impact of the chemotherapeutic drugs (individual cumulative doses) received before transplant on stem-cell yield and hematologic recovery was investigated. Univariate analysis failed to identify any variable that significantly affected progenitor-cell content., Results: Regarding the impact of pre-transplant chemotherapy on early engraftment, only cumulative doses of cytarabine (r=0.28, p=0.04) and cisplatin (r=0.32,p=0.02) had a negative influence on neutrophil recovery (to >0.5x10(9)/L), but the significance of this was not maintained in multivariate analysis. We did not find any chemotherapeutic drug that negatively affected platelet recovery (to >20x10(9)/L). By contrast, administration of several drugs, including doxorubicin, procarbazine, nitrogen mustard, cytarabine and cisplatin, significantly delayed complete trilineage reconstitution. In multivariate analysis, only previous doxorubicin retained statistical significance (p=0.014)., Interpretation and Conclusions: Our results show that pre-transplant chemotherapy has little or no influence on progenitor-cell yield and short-term engraftment after ABMT. In contrast, we found that cumulative doxorubicin doses have an independent influence on long-term engraftment. In heavily pretreated lymphoma patients in whom poor PBPC mobilization is expected, BMT may represent an attractive option.

Background and Objectives: Whether human mesenchymal stem cells (MSC) can be transplanted is controversial and their presence in peripheral blood is not fully accepted. In the present study we have analyzed whether, within the allogeneic transplantation setting, MSC are of host or donor origin., Design and Methods: Bone marrow MSC from 19 patients who had undergone allogeneic transplantation were expanded and identified using immunophenotypic markers. After that, chimerism studies were performed using reverse transcription polymerase chain reaction of short tandem repeat (STR) loci. Analyses were carried out at different time-points after transplantation, with a total of 44 samples studied. Bone marrow was used as the source of stem cells for transplantation in 4 cases and peripheral blood in 15 cases. The conditioning regimen was standard in 9 patients and non-myeloablative in 10 patients., Results: Our results show that in the great majority of cases analyzed (17 out 19), MSC were of host origin. However, in 2 patients with multiple myeloma who had received a reduced intensity transplantation using peripheral blood stem cells, MSC were partially of donor origin (60.17% and 26.13% of total MSC)., Interpretation and Conclusions: These findings indicate that after allogeneic transplantation MSC from the donor can engraft in bone marrow. Moreover, since the stem cells were obtained from peripheral blood, it can be concluded that MSC circulate among mobilized peripheral blood stem cells and can engraft in bone marrow after allogeneic transplantation.

The present work analyzes the hematopoietic progenitor cells (HPC) in myelodysplastic syndrome (MDS) patients using both an immunophenotypical and a functional approaches in order to know whether they are similar in patients with or without cytogenetic abnormalities. Among CD34+ HPC, the proportion of myeloid committed progenitors was higher in patients with an abnormal karyotype. Ninety MDS patients were studied. Patients with abnormal karyotype showed a similar platting efficiency than patients with normal cytogenetics. Trisomy 8 and 5q- showed a significant higher P.E. than patients with normal karyotype or monosomy 7. We observed that when the most immature HPC were studied, the total number of granulo-monocytic colonies produced by LTBMC was higher in the normal karyotype group. In summary, the present study shows that in MDS the HPC are impaired; this impairment is deeper in patients with abnormal karyotype.

Aim: Experimental animal studies suggest that the use of skeletal myoblast in patients with myocardial infarction may result in improved cardiac function. The aim of the study was to assess the feasibility and safety of this therapy in patients with myocardial infarction., Methods and Results: Twelve patients with old myocardial infarction and ischaemic coronary artery disease underwent treatment with coronary artery bypass surgery and intramyocardial injection of autologous skeletal myoblasts obtained from a muscle biopsy of vastus lateralis and cultured with autologous serum for 3 weeks. Global and regional cardiac function was assessed by 2D and ABD echocardiogram. 18F-FDG and 13N-ammonia PET studies were used to determine perfusion and viability. Left ventricular ejection fraction (LVEF) improved from 35.5+/-2.3% before surgery to 53.5+/-4.98% at 3 months (P=0.002). Echocardiography revealed a marked improvement in regional contractility in those cardiac segments treated with skeletal myoblast (wall motion score index 2.64+/-0.13 at baseline vs 1.64+/-0.16 at 3 months P=0.0001). Quantitative 18F-FDG PET studies showed a significant (P=0.012) increased in cardiac viability in the infarct zone 3 months after surgery. No statistically significant differences were found in 13N-ammonia PET studies. Skeletal myoblast implant was not associated with an increase in adverse events. No cardiac arrhythmias were detected during early follow-up., Conclusions: In patients with old myocardial infarction, treatment with skeletal myoblast in conjunction with coronary artery bypass is safe and feasible and is associated with an increased global and regional left ventricular function,improvement in the viability of cardiac tissue in the infarct area and no induction of arrhythmias.

Purpose: To analyze, in two-stages long term bone marrow cultures (LTBMC), the efficiency of the method used for irradiation of the stroma and to check if after that, it's capable to support the hematopoiesis., Material and Methods: We have used for the first inoculum, bone marrow cells from eight controls. They were cultured at 2 x 10(6) cells/ml concentration until obtaining a fully confluent stroma which was irradiated with 15 Gy. After that, over this layer, we put a second inoculum with bone marrow cells in four cases, and with peripheral blood cells in four others, at a concentration of 1 x 10(5) cells/mL. In four cases the first inoculum was from a man and the second from a woman. The culture's haematopoietic activity was determined by the number of CFU-GM, total cell counts and the differential counts during four weeks. At the end of the study we recovered the cells from the supernatant of the culture and they were analyzed by in situ hybridization with an alpha centromeric probe specific to the X chromosome., Results: In all cases we obtained a confluent stroma with production of haematopoiesis (cobblestone areas). The cells recovered at the end of the culture were, in all cases, from the second inoculum because they had a double signal with the X probe. The number of CFU-GM obtained was higher in bone marrow than in peripheral blood (34.5 and 9.2 respectively) however, the total cells were similar in both cases (2.3 x 10(5) and 2.9 x 10(5)) although the cellular subtypes varied depending on the second inoculum (B.M. or P.B.)., Conclusion: Our data confirm that the dose of 15 Gy eradicate the haemopoiesis of first inoculum, which allowed to analyze stroma and progenitor cells, separately. In addition, the stroma is capable to support the hematopoiesis generated by progenitor cells from peripheral and bone marrow.