PS1/γ-Secretase-Mediated Cadherin Cleavage Induces ß-Catenin Nuclear Translocation and Osteogenic Differentiation of Human Bone Marrow Stromal Cells.

Bone marrow stromal cells (BMSCs) are considered a promising tool for bone bioengineering. However, the mechanisms controlling osteoblastic commitment are still unclear. Osteogenic differentiation of BMSCs requires the activation of ß-catenin signaling, classically known to be regulated by the canonical Wnt pathway. However, BMSCs treatment with canonical Wnts in vitro does not always result in osteogenic differentiation and evidence indicates that a more complex signaling pathway, involving cadherins, would be required to induce ß-catenin signaling in these cells. Here we showed that Wnt3a alone did not induce TCF activation in BMSCs, maintaining the cells at a proliferative state. On the other hand, we verified that, upon BMSCs osteoinduction with dexamethasone, cadherins were cleaved by the PS1/γ-secretase complex at the plasma membrane, and this event was associated with an enhanced ß-catenin translocation to the nucleus and signaling. When PS1/γ-secretase activity was inhibited, the osteogenic process was impaired. Altogether, we provide evidence that PS1/γ-secretase-mediated cadherin cleavage has as an important role in controlling ß-catenin signaling during the onset of BMSCs osteogenic differentiation, as part of a complex signaling pathway responsible for cell fate decision. A comprehensive map of these pathways might contribute to the development of strategies to improve bone repair.

The effect of autologous concentrated bone-marrow grafting on the healing of femoral shaft non-unions after locked intramedullary nailing.

The aim of this study was to assess the union rates in a series of patients with failed femoral shaft aseptic non-union who were treated with percutaneous concentrated autologous bone marrow grafting. Bone marrow harvesting and cell injection were performed under general anaesthesia in a single surgical procedure. Radiographic union was diagnosed in fractures with a score ≥ 10 according to the radiographic union scale in tibial fractures (RUST) and confirmed by clinical examination. Eight out of 16 patients progressed to consolidation (RUST score ≥ 10). Radiographic evidence of fracture union was observed at an average of 4.75 ± 1.75 months (range 3 to 8 months). All eight patients who did not progress to union within 12 months following the cell grafting procedure had a RUST score ≤ 10 (range 4 to 9). There were no differences in age, number of previous surgeries, duration of nonunion and preoperative RUST score between the patients that developed solid union and those with failed consolidation. However, a relationship between the number of osteoprogenitors injected and the rate of union was noted, 20.2 ± 8.6 × 10(8) versus 9.8 ± 4.3 × 10(8), p<0.005, between the patients with and without union, respectively. The efficacy of percutaneous autologous concentrated bone marrow grafting seems to be related to the number of osteoprogenitors available in the aspirates. Optimisation of the aspiration technique and concentration process is of paramount importance to increase the incidence of a successful outcome.

Interaction between IL-6 and TNF-α genotypes associated with bacteremia in multiple myeloma patients submitted to autologous stem cell transplantation (ASCT).

Stem cell transplantation affects patient׳s vulnerability to infections due to immunological changes related to chemotherapy. Multiple myeloma is characterized by susceptibility to infections, and IL-6 and TNF-α increased levels affect immune response (IR). Polymorphisms in promoter region of cytokine genes may alter expression levels and affect IR. We performed interaction analysis of IL-6 (-174G/C) and TNF-α (-308G/A) polymorphisms with infection susceptibility in 148 patients classified accordingly to infection status and found an interaction when compared groups with and without bacteremia (p=0.0380). The interaction may be more important than single effects for the IR associated with the infection susceptibility in ASCT.

Cryopreservation of peripheral blood stem cell: the influence of cell concentration on cellular and hematopoietic recovery.

BACKGROUND: The optimal cryopreservation cell concentration during the peripheral blood stem cell (PBSC) collection is a controversial topic. We evaluated the influence of cryopreservation concentration on the recovery of hematopoietic progenitor cells and the kinetics of hematopoietic recovery of autologous stem cell transplant patients. STUDY DESIGN AND METHODS: In this retrospective study, we compared two different cryopreservation protocols: 1×10(8) cells/mL (Protocol A) and 2×10(8) cells/mL (Protocol B). A total of 419 PBSCs were analyzed with regard to the number of viable cells and colony-forming units-granulocytes-monocytes (CFU-GM) progenitors. The hematopoietic recovery of 275 patients who received PBSCs cryopreserved at a dose of 1×10(8) cells/mL (Group A) and 2×10(8) cells/mL (Group B) were compared. RESULTS: There were no significant differences in recovery of viable cells between Protocol A and Protocol B. The median of recovery of CFU-GM progenitors was significantly higher in Protocol B (41.2 vs. 57.3, p<0.01). The median times to neutrophil recovery (≥500×10(6) /L) and platelet (PLT) recovery (≥20×10(9) /L) in Groups A and B were 11 days versus 11 days and 12 days versus 12 days, respectively. However, by Kaplan and Meier analyses, Group B recovered neutrophils with a little delay (p=0.01). No difference was observed with regard to time to PLT recovery. On multivariate analysis, we found that the number of CD34+ cells and CFU-GM progenitors had a significant influence on hematopoietic recovery. CONCLUSION: Cryopreservation of PBSCs at a dose of 2×10(8) cells/mL did not affect the recovery rate of viable cells or the hematopoietic recovery of autologous stem cell transplant patients.

Osteoblasts and bone marrow mesenchymal stromal cells control hematopoietic stem cell migration and proliferation in 3D in vitro model.

BACKGROUND: Migration, proliferation, and differentiation of hematopoietic stem cells (HSCs) are dependent upon a complex three-dimensional (3D) bone marrow microenvironment. Although osteoblasts control the HSC pool, the subendosteal niche is complex and its cellular composition and the role of each cell population in HSC fate have not been established. In vivo models are complex and involve subtle species-specific differences, while bidimensional cultures do not reflect the 3D tissue organization. The aim of this study was to investigate in vitro the role of human bone marrow-derived mesenchymal stromal cells (BMSC) and active osteoblasts in control of migration, lodgment, and proliferation of HSCs. METHODOLOGY/PRINCIPAL FINDINGS: A complex mixed multicellular spheroid in vitro model was developed with human BMSC, undifferentiated or induced for one week into osteoblasts. A clear limit between the two stromal cells was established, and deposition of extracellular matrix proteins fibronectin, collagens I and IV, laminin, and osteopontin was similar to the observed in vivo. Noninduced BMSC cultured as spheroid expressed higher levels of mRNA for the chemokine CXCL12, and the growth factors Wnt5a and Kit ligand. Cord blood and bone marrow CD34(+) cells moved in and out the spheroids, and some lodged at the interface of the two stromal cells. Myeloid colony-forming cells were maintained after seven days of coculture with mixed spheroids, and the frequency of cycling CD34(+) cells was decreased. CONCLUSIONS/SIGNIFICANCE: Undifferentiated and one-week osteo-induced BMSC self-assembled in a 3D spheroid and formed a microenvironment that is informative for hematopoietic progenitor cells, allowing their lodgment and controlling their proliferation.

Macrophage migration inhibitory factor is critical to interleukin-5-driven eosinophilopoiesis and tissue eosinophilia triggered by Schistosoma mansoni infection.

Macrophage migration inhibitory factor (MIF) participates in the pathogenesis of inflammatory diseases, including asthma, in which it enhances airway hypersensitivity and tissue eosinophilia. Herein, we investigated the role of MIF in eosinophilopoiesis and tissue eosinophilia using Schistosoma mansoni infection. MIF-deficient (Mif(-/-)) mice had similar numbers of adult worms, eggs, and granulomas compared to wild-type mice, but the size of granulomas was strikingly reduced due to smaller numbers of eosinophils. MIF did not affect the acquired response to infection, as Mif(-/-) mice produced normal amounts of Th2 cytokines and IgE. Nevertheless, recombinant MIF (rMIF) behaved as a chemoattractant for eosinophils, what could partially explain the reduced eosinophilia in infected Mif(-/-) mice. Moreover, the percentage of eosinophils was reduced in bone marrows of Mif(-/-) mice chronically infected with S. mansoni compared to wild type. Mif(-/-) had impaired eosinophilopoiesis in response to interleukin (IL)-5 and addition of rMIF to bone marrow cultures from IL-5 transgenic mice enhanced the generation of eosinophils. In the absence of MIF, eosinophil precursors were unable to survive the IL-5-supplemented cell culture, and were ingested by macrophages. Treatment with pancaspase inhibitor z-VAD or rMIF promoted the survival of eosinophil progenitors. Together, these results indicate that MIF participates in IL-5-driven maturation of eosinophils and in tissue eosinophilia associated with S. mansoni infection.

Transendocardial autologous bone marrow mononuclear cell injection in ischemic heart failure: postmortem anatomicopathologic and immunohistochemical findings.

BACKGROUND: Cell-based therapies for treatment of ischemic heart disease are currently under investigation. We previously reported the results of a phase I trial of transendocardial injection of autologous bone marrow mononuclear (ABMM) cells in patients with end-stage ischemic heart disease. The current report focuses on postmortem cardiac findings from one of the treated patients, who died 11 months after cell therapy. METHODS AND RESULTS: Anatomicopathologic, morphometric, and immunocytochemical findings from the anterolateral ventricular wall (with cell therapy) were compared with findings from the interventricular septum (normal perfusion and no cell therapy) and from the inferoposterior ventricular wall (extensive scar tissue and no cell therapy). No signs of adverse events were found in the cell-injected areas. Capillary density was significantly higher (P<0.001) in the anterolateral wall than in the previously infarcted tissue in the posterior wall. The prominent vasculature of the anterolateral wall was associated with hyperplasia of pericytes, mural cells, and adventitia. Some of these cells had acquired cytoskeletal elements and contractile proteins (troponin, sarcomeric alpha-actinin, actinin), as well as the morphology of cardiomyocytes, and appeared to have migrated toward adjacent bundles of cardiomyocytes. CONCLUSIONS: Eleven months after treatment, morphological and immunocytochemical analysis of the sites of ABMM cell injection showed no abnormal cell growth or tissue lesions and suggested that an active process of angiogenesis was present in both the fibrotic cicatricial tissue and the adjacent cardiac muscle. Some of the pericytes had acquired the morphology of cardiomyocytes, suggesting long-term sequential regeneration of the cardiac vascular tree and muscle.

Transendocardial, autologous bone marrow cell transplantation for severe, chronic ischemic heart failure.

BACKGROUND: This study evaluated the hypothesis that transendocardial injections of autologous mononuclear bone marrow cells in patients with end-stage ischemic heart disease could safely promote neovascularization and improve perfusion and myocardial contractility. METHODS AND RESULTS: Twenty-one patients were enrolled in this prospective, nonrandomized, open-label study (first 14 patients, treatment; last 7 patients, control). Baseline evaluations included complete clinical and laboratory evaluations, exercise stress (ramp treadmill), 2D Doppler echocardiogram, single-photon emission computed tomography perfusion scan, and 24-hour Holter monitoring. Bone marrow mononuclear cells were harvested, isolated, washed, and resuspended in saline for injection by NOGA catheter (15 injections of 0.2 cc). Electromechanical mapping was used to identify viable myocardium (unipolar voltage > or =6.9 mV) for treatment. Treated and control patients underwent 2-month noninvasive follow-up, and treated patients alone underwent a 4-month invasive follow-up according to standard protocols and with the same procedures used as at baseline. Patient population demographics and exercise test variables did not differ significantly between the treatment and control groups; only serum creatinine and brain natriuretic peptide levels varied in laboratory evaluations at follow-up, being relatively higher in control patients. At 2 months, there was a significant reduction in total reversible defect and improvement in global left ventricular function within the treatment group and between the treatment and control groups (P=0.02) on quantitative single-photon emission computed tomography analysis. At 4 months, there was improvement in ejection fraction from a baseline of 20% to 29% (P=0.003) and a reduction in end-systolic volume (P=0.03) in the treated patients. Electromechanical mapping revealed significant mechanical improvement of the injected segments (P<0.0005) at 4 months after treatment. CONCLUSIONS: Thus, the present study demonstrates the relative safety of intramyocardial injections of bone marrow-derived stem cells in humans with severe heart failure and the potential for improving myocardial blood flow with associated enhancement of regional and global left ventricular function.

Myelopoiesis in the omentum of normal mice and during abdominal inflammatory processes.

Coelomic cavities are relatively isolated from the systemic circulation of blood cells. Resident cell populations have a proper phenotype and kinetics, maintaining their steady-state populations and their responsiveness to local inflammatory reactions, in which the number and quality of coelomic cells can be greatly increased and modified. We have addressed the question of whether the increase in cell infiltrate in the inflamed abdominal cavity is sustained by the proliferation of myeloid cells in the omentum, and if so what are the characteristics of the progenitor cells involved and how the omentum controls their proliferation and differentiation. In the omentum under normal conditions and with inflammation due to schistosomal infection we found that pluripotent early myeloid progenitors were capable of giving rise to all the myeloid lineages in clonogenic assays, but not to the totipotent blood stem cells. Besides the major haemopoietins (GM-CSF, M-CSF, G-CSF, IL-5), the omentum stroma constitutively expressed SDF-1 alpha, the chemokine which elicits homing of circulating early haemopoietic progenitors. While normal omentum stroma produced LIF, its expression was substituted by SCF in inflamed tissues. In the first situation a slow steady-state renewal of progenitors is potentially favoured, while their intense expansion may be predominant in the latter one. We propose that the increase in cells in the abdominal cavity in inflammatory reactions is due to the enhanced input and expansion of early myeloid progenitors sustaining the in situ production of abdominal cell populations, rather than to the input of systemic circulating inflammatory cells.