Hélio S. Dutra

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 ≥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.

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 &agr;-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.

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.

In vitro induction of the fat-storing phenotype in a liver connective tissue cell line-GRX

SummaryLiver connective tissue cells have been characterized as perisinusoidal myofibroblasts and hepatic lipocytes (Ito cells, fat-storing cells). A concept of a single mesenchymal cell population that may be modulated between these two phenotypes has been postulated. We have previously established a continuous murine cell line, GRX, obtained from fibrotic granulomatous lesions induced by schistosomal infection in mouse liver. This cell line is considered to represent liver myofibroblasts. In the present study we have induced the conversion of these cells into lipocyte (fat storing) phenotype by treatment with insulin and indomethacin. We have quantified the lipid synthesis and the increase of activity of involved enzymes during the induction of the fat-storing phenotype and described modifications of cell organization along this modulation of cell functions.

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.

Multicellular spheroids of bone marrow stromal cells: a three-dimensional in vitro culture system for the study of hematopoietic cell migration

Cell fate decisions are governed by a complex interplay between cell-autonomous signals and stimuli from the surrounding tissue. In vivo cells are connected to their neighbors and to the extracellular matrix forming a complex three-dimensional (3-D) microenvironment that is not reproduced in conventional in vitro systems. A large body of evidence indicates that mechanical tension applied to the cytoskeleton controls cell proliferation, differentiation and migration, suggesting that 3-D in vitro culture systems that mimic the in vivo situation would reveal biological subtleties. In hematopoietic tissues, the microenvironment plays a crucial role in stem and progenitor cell survival, differentiation, proliferation, and migration. In adults, hematopoiesis takes place inside the bone marrow cavity where hematopoietic cells are intimately associated with a specialized three 3-D scaffold of stromal cell surfaces and extracellular matrix that comprise specific niches. The relationship between hematopoietic cells and their niches is highly dynamic. Under steady-state conditions, hematopoietic cells migrate within the marrow cavity and circulate in the bloodstream. The mechanisms underlying hematopoietic stem/progenitor cell homing and mobilization have been studied in animal models, since conventional two-dimensional (2-D) bone marrow cell cultures do not reproduce the complex 3-D environment. In this review, we will highlight some of the mechanisms controlling hematopoietic cell migration and 3-D culture systems.

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

Abstract. 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-1α, 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.

Haematopoietic capacity of colony-forming cells mobilized in hepatic inflammatory reactions as compared to that of normal bone marrow cells.

Chronic inflammatory periovular granulomatous reactions elicited in liver by schistosomal infection are a site of active myelopoiesis. We quantified the colony-forming cells (CFCs) in granulomas and found that the whole liver contains a number of CFCs roughly equivalent to 50% of a femur. Clonogenic analysis showed the presence of committed as well as pluripotent and totipotent CFCs. Long-term Dexter-type cultures showed that the granuloma-derived totipotent CFCs do not have self-renewal capacity. Hence, they did not correspond functionally to haematopoietic stem cells, despite the fact that the stroma established by adherent cells harvested from granulomas had the capacity to sustain long-term proliferation of bone-marrow-derived haematopoietic stem cells. We conclude that myelopoietic cytokines produced by inflammatory reactions in schistosomiasis elicit mobilization of bone marrow CFCs into the circulation, which can settle in hepatic granulomas. This environment may induce their proliferation and differentiation, but not their self-renewal, sustaining temporary production of myeloid cell lineages which nevertheless depends upon cell renewal from the bone marrow pool of haematopoietic precursors.

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.

Murine schistosomiasis mansoni: experimental analysis of bone marrow and peripheral myelopoiesis.

Abstract In schistosomiasis a systemic hyperplasia of the monomacrophagic cell lineage is associated with its mild modifications in myelograms and hemograms. We monitored the in vitro proliferation of myeloid precursors obtained from bone marrow, blood, spleen, and liver. The macrophage colony-forming unit (M-CFU) numbers were stable in bone marrow but increased progressively in spleen and in liver, reaching in each organ the values equivalent to one femur. The bone marrow had an increased production and enhanced capacity to release M-CFU. Their quantitative increase in blood and in peripheral tissues of schistosome-infected mice was associated with their qualitative modifications: augmented proliferative capacity, enhanced adhesion, and accelerated differentiation. The accelerated release of monomacrophage progenitors and their enhanced proliferation in peripheral tissues potentially account for the relatively low involvement of the bone marrow and for an efficient in situ production of phagocytes, which participate in host reactions to parasites.