Increased Levels of NOTCH1, NF-kappa B, and Other Interconnected Transcription Factors Characterize Primitive Sets of Hematopoietic Stem Cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Isolation and characterization of Wharton's jelly-derived multipotent mesenchymal stromal cells obtained from bovine umbilical cord and maintained in a defined serum-free three-dimensional system

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Role of natural killer cells in antitumor resistance

Natural killer cells constitute a population of lymphocytes able to non-specifically destroy virus-infected and some kinds of tumor cells. Since this lytic activity was shown by non-immunized animals the phenomenon is denominated natural killer (NK) activity and contrasts with specific cytotoxicity performed by cytolytic T lymphocytes (CTLs) because it does not depends on MHC-restricted peptides recognition. In fact, the main feature of most functional receptors of NK cells (NKRs) is their ability to be inhibited by different kinds of class I MHC antigens. In the middle of the 1950's, Burnet & Thomas forged the concept of tumor immunosurveillance and NK cells can be considered one of the main figures in this phenomenon both for effector and regulatory functions. In the present review the early studies on the biology of NK cells were revisited and both their antitumor activity and dependence on the activation by cytokines are discussed.

Interaction between mesenchymal stem cells and Ti-30Ta alloy after surface treatment

In this study, in vitro cytocompatibility was investigated in the Ti-30Ta alloy after two kinds of surfaces treatments: alkaline and biomimetic treatment. Each condition was evaluated by scanning electron microscopy/energy-dispersive X-ray spectroscopy. Cellular adhesion, viability, protein expression, morphology, and differentiation were evaluated with Bone marrow stromal cells (MSCs) to investigate the short and long-term cellular response by fluorescence microscope imaging and colorimetric assays techniques. Two treatments exhibited similar results with respect to total protein content and enzyme activity as compared with alloy without treatment. However, it was observed improved of the biomineralization, bone matrix formation, enzyme activity, and MSCs functionality after biomimetic treatment. These results indicate that the biomimetic surface treatment has a high potential for enhanced osseointegration. © 2013 Wiley Periodicals, Inc.

Comparação entre enxerto ósseo autógeno e rhMP-2 (infuse Bone Graft) na reconstrução de maxila atrófica anterior

Pós-graduação em Odontologia - FOAR

Do progenitor cells from different tissue have the same phenotype?

The purpose of this work was to validate isolation methods for sheep mesenchymal stem cells (MSC) from different sources and to explore the hypothesis that MSC exhibit markers of the same phenotype independent from tissue source. Cells derived from ovine bone marrow, synovial membrane and adipose tissue were characterized using the following markers: CD44, CD45, CD11b and MHC-I. The isolated MSC were cultivated, went through osteogenic, chondrogenic and adipogenic differentiation, and were characterized by flow cytometry using mouse anti-ovine CD44, CD45 and MHC-I monoclonal antibody (mAb), and mouse anti-bovine CD11b mAb. Ovine MSC from all three sources differentiated under chondorgenic, osteogenic and adipogenic conditions. Also, MSC from the three tissues were found to express CD44 and MHC-I but lack of CD11b and CD45. The results obtained revealed that our isolation methods for the different tissues tested are valid and that MSC from the three sources studied have same immunophenotic characteristics. (C) 2014 Published by Elsevier Ltd. All rights reserved.

Mesenchymal stem cells modulate release of matrix proteins from tendon surfaces in vitro: a potential beneficial therapeutic effect

Aim: Injury of tendons contained within a synovial environment, such as joint, bursa or tendon sheath, frequently fails to heal and releases matrix proteins into the synovial fluid, driving inflammation. This study investigated the effectiveness of cells to seal tendon surfaces and provoke matrix synthesis as a possible effective injectable therapy. Materials & methods: Equine flexor tendon explants were cultured overnight in suspensions of bone marrow and synovium-derived mesenchymal stems cells and, as controls, two sources of fibroblasts, derived from tendon and skin, which adhered to the explants. Release of the most abundant tendon extracellular matrix proteins into the media was assayed, along with specific matrix proteins synthesis by real-time PCR. Results: Release of extracellular matrix proteins was influenced by the coating cell type. Fibroblasts from skin and tendon appeared less capable of preventing the release of matrix proteins than mesenchymal stems cells. Conclusion: The source of cell is an important consideration for cell therapy.

M-CSF Priming of Osteoclast Precursors Can Cause Osteoclastogenesis-Insensitivity, Which Can be Prevented and Overcome on Bone

Osteoclasts and macrophages share progenitors that must receive decisive lineage signals driving them into their respective differentiation routes. Macrophage colony stimulation factor M-CSF is a common factor; bone is likely the stimulus for osteoclast differentiation. To elucidate the effect of both, shared mouse bone marrow precursor myeloid blast was pre-cultured with M-CSF on plastic and on bone. M-CSF priming prior to stimulation with M-CSF and osteoclast differentiation factor RANKL resulted in a complete loss of osteoclastogenic potential without bone. Such M-CSF primed cells expressed the receptor RANK, but lacked the crucial osteoclastogenic transcription factor NFATc1. This coincided with a steeply decreased expression of osteoclast genes TRACP and DC-STAMP, but an increased expression of the macrophage markers F4/80 and CD11b. Compellingly, M-CSF priming on bone accelerated the osteoclastogenic potential: M-CSF primed cells that had received only one day M-CSF and RANKL and were grown on bone already expressed an array of genes that are associated with osteoclast differentiation and these cells differentiated into osteoclasts within 2 days. Osteoclastogenesis-insensitive precursors grown in the absence of bone regained their osteoclastogenic potential when transferred to bone. This implies that adhesion to bone dictates the fate of osteoclast precursors. Common macrophage-osteoclast precursors may become insensitive to differentiate into osteoclasts and regain osteoclastogenesis when bound to bone or when in the vicinity of bone. J. Cell. Physiol. 229: 210-225, 2014. (c) 2014 Wiley Periodicals, Inc.

Diabetes reduces mesenchymal stem cells in fracture healing through a TNF alpha-mediated mechanism

Diabetes interferes with bone formation and impairs fracture healing, an important complication in humans and animal models. The aim of this study was to examine the impact of diabetes on mesenchymal stem cells (MSCs) during fracture repair.Fracture of the long bones was induced in a streptozotocin-induced type 1 diabetic mouse model with or without insulin or a specific TNF alpha inhibitor, pegsunercept. MSCs were detected with cluster designation-271 (also known as p75 neurotrophin receptor) or stem cell antigen-1 (Sca-1) antibodies in areas of new endochondral bone formation in the calluses. MSC apoptosis was measured by TUNEL assay and proliferation was measured by Ki67 antibody. In vitro apoptosis and proliferation were examined in C3H10T1/2 and human-bone-marrow-derived MSCs following transfection with FOXO1 small interfering (si)RNA.Diabetes significantly increased TNF alpha levels and reduced MSC numbers in new bone area. MSC numbers were restored to normal levels with insulin or pegsunercept treatment. Inhibition of TNF alpha significantly reduced MSC loss by increasing MSC proliferation and decreasing MSC apoptosis in diabetic animals, but had no effect on MSCs in normoglycaemic animals. In vitro experiments established that TNF alpha alone was sufficient to induce apoptosis and inhibit proliferation of MSCs. Furthermore, silencing forkhead box protein O1 (FOXO1) prevented TNF alpha-induced MSC apoptosis and reduced proliferation by regulating apoptotic and cell cycle genes.Diabetes-enhanced TNF alpha significantly reduced MSC numbers in new bone areas during fracture healing. Mechanistically, diabetes-enhanced TNF alpha reduced MSC proliferation and increased MSC apoptosis. Reducing the activity of TNF alpha in vivo may help to preserve endogenous MSCs and maximise regenerative potential in diabetic patients.

Human immature dental pulp stem cells share key characteristic features with limbal stem cells

Objectives: Limbal stem cells (LSC) are self-renewing, highly proliferative cells in vitro, which express a set of specific markers and in vivo have the capacity to reconstruct the entire corneal epithelium in cases of ocular surface injury. Currently, LSC transplantation is a commonly used procedure in patients with either uni- or bilateral total limbal stem cells deficiency (TLSCD). Although LSC transplantation holds great promise for patients, several problems need to be overcome. In order to find an alternative source of cells that can partially substitute LSC in cornea epithelium reconstruction, we aimed at investigating whether human immature dental pulp stem cells (hIDPSC) would present similar key characteristics as LSC and whether they could be used for corneal surface reconstruction in a rabbit TLSCD model. Materials: We used hIDPSC, which co-express mesenchymal and embryonic stem cell markers and present the capacity to differentiate into derivative cells of the three germinal layers. TLSCD was induced by chemical burn in one eye of rabbits. After 30 days, the opaque tissue formed was removed by superficial keratectomy. Experimental group received undifferentiated hIDPSC, while control group only received amniotic membrane (AM). Both groups were sacrificed after 3 months. Results and conclusions: We have demonstrated, using immunohistochemistry and reverse transcription-polymerase chain reaction, that hIDPSCs express markers in common with LSC, such as ABCG2, integrin beta 1, vimentin, p63, connexin 43 and cytokeratins 3/12. They were also capable of reconstructing the eye surface after induction of unilateral TLSCD in rabbits, as shown by morphological and immunohistochemical analysis using human-specific antibodies against limbal and corneal epithelium. Our data suggest that hIDPSCs share similar characteristics with LSC and might be used as a potential alternative source of cells for corneal reconstruction.

IGFBP7 participates in the reciprocal interaction between acute lymphoblastic leukemia and BM stromal cells and in leukemia resistance to asparaginase

The interaction of acute lymphoblastic leukemia (ALL) blasts with bone marrow (BM) stromal cells (BMSCs) has a positive impact on ALL resistance to chemotherapy. We investigated the modulation of a series of putative asparaginase-resistance/sensitivity genes in B-precursor ALL cells upon coculture with BMSCs. Coculture with stromal cells resulted in increased insulin-like growth factor (IGF)-binding protein 7 (IGFBP7) expression by ALL cells. Assays with IGFBP7 knockdown ALL and stromal cell lines, or with addition of recombinant rIGFBP7 (rIGFBP7) to the culture medium, showed that IGFBP7 acts as a positive regulator of ALL and stromal cells growth, and significantly enhances in-vitro resistance of ALL to asparaginase. In these assays, IGFBP7 function occurred mainly in an insulin-and stromal-dependent manner. ALL cells were found to contribute substantially to extracellular IGFBP7 levels in the conditioned coculture medium. Diagnostic BM plasma from children with ALL had higher levels of IGFBP7 than controls. IGFBP7, in an insulin/IGF-dependent manner, enhanced asparagine synthetase expression and asparagine secretion by BMSCs, thus providing a stromal-dependent mechanism by which IGFBP7 protects ALL cells against asparaginase in this coculture system. Importantly, higher IGFBP7 mRNA levels were associated with lower leukemia-free survival (Cox regression model, P = 0.003) in precursor B-cell Ph(-) ALL patients (n = 147) treated with a contemporary polychemotherapy protocol.

Muscle reorganisation through local injection of stem cells in the diaphragm of mdx mice

Background: The diaphragm is the major respiratory muscle affected by Duchenne muscular dystrophy (DMD) and is responsible for causing 80% of deaths. The use of mechanical forces that act on the body or intermittent pressure on the airways improves the quality of life of patients but does not prevent the progression of respiratory failure. Thus, diseases that require tissue repair, such as DMD, represent a group of pathologies that have great potential for cell therapy. The application of stem cells directly into the diaphragm instead of systemic application can reduce cell migration to other affected areas and increase the chances of muscle reorganisation. The mdx mouse is a suitable animal model for this research because its diaphragmatic phenotype is similar to human DMD. Therefore, the aim of this study was to assess the potential cell implantation in the diaphragm muscle after the xenotransplantation of stem cells. Methods: A total of 9 mice, including 3 control BALB/Cmice, 3 5-month-old mdx mice without stem cell injections and 3 mdx mice injected with stem cells, were used. The animals injected with stem cells underwent laparoscopy so that stem cells from GFP-labelled rabbit olfactory epithelium could be locally injected into the diaphragm muscle. After 8 days, all animals were euthanised, and the diaphragm muscle was dissected and subjected to histological and immunohistochemical analyses. Results: Both the fresh diaphragm tissue and immunohistochemical analyses showed immunopositive GFP labelling of some of the cells and immunonegativity of myoblast bundles. In the histological analysis, we observed a reduction in the inflammatory infiltrate as well as the presence of a few peripheral nuclei and myoblast bundles. Conclusion: We were able to implant stem cells into the diaphragm via local injection, which promoted moderate muscle reorganisation. The presence of myoblast bundles cannot be attributed to stem cell incorporation because there was no immunopositive labelling in this structure. It is believed that the formation of the bundles may have been stimulated by cellular signalling mechanisms that have not yet been elucidated.

Antiproliferative Effects of Fluoxetine on Colon Cancer Cells and in a Colonic Carcinogen Mouse Model

The antidepressant fluoxetine has been under discussion because of its potential influence on cancer risk. It was found to inhibit the development of carcinogen-induced preneoplastic lesions in colon tissue, but the mechanisms of action are not well understood. Therefore, we investigated anti-proliferative effects, and used HT29 colon tumor cells in vitro, as well as C57BL/6 mice exposed to intra-rectal treatment with the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as models. Fluoxetine increased the percentage of HT29 cells in the G(0)/G(1) phase of cell-cycle, and the expression of p27 protein. This was not related to an induction of apoptosis, reactive oxygen species or DNA damage. In vivo, fluoxetine reduced the development of MNNG-induced dysplasia and vascularization-related dysplasia in colon tissue, which was analyzed by histopathological techniques. An anti-proliferative potential of fluoxetine was observed in epithelial and stromal areas. It was accompanied by a reduction of VEGF expression and of the number of cells with angiogenic potential, such as CD133, CD34, and CD31-positive cell clusters. Taken together, our findings suggest that fluoxetine treatment targets steps of early colon carcinogenesis. This confirms its protective potential, explaining at least partially the lower colon cancer risk under antidepressant therapy.

Hedgehog signaling and osteoblast gene expression are regulated by purmorphamine in human mesenchymal stem cells

Several biological events are controlled by Hedgehog (Hh) signaling, including osteoblast phenotype development. This study aimed at evaluating the gene expression profile of human mesenchymal stem cells (hMSCs) treated with the Hh agonist, purmorphamine, focusing on Hh signaling and osteoblast differentiation. hMSCs from bone marrow were cultured in non-osteogenic medium with or without purmorphamine (2 mu M) for periods of up to 14 days. Purmorphamine up-regulated gene expression of the mediators of Hh pathway, SMO, PTCH1, GLI1, and GLI2. The activation of Hh pathway by purmorphamine increased the expression of several genes (e.g., RUNX2 and BMPs) related to osteogenesis. Our results indicated that purmorphamine triggers Hh signaling pathway in hMSCs, inducing an increase in the expression of a set of genes involved in the osteoblast differentiation program. Thus, we conclude that Hh is a crucial pathway in the commitment of undifferentiated cells to the osteoblast lineage. J. Cell. Biochem. 113: 204208, 2012. (C) 2011 Wiley Periodicals, Inc.

Multiple Intravenous Administrations of Human Umbilical Cord Blood Cells Benefit in a Mouse Model of ALS

Background: A promising therapeutic strategy for amyotrophic lateral sclerosis (ALS) is the use of cell-based therapies that can protect motor neurons and thereby retard disease progression. We recently showed that a single large dose (25x10(6) cells) of mononuclear cells from human umbilical cord blood (MNC hUCB) administered intravenously to pre-symptomatic G93A SOD1 mice is optimal in delaying disease progression and increasing lifespan. However, this single high cell dose is impractical for clinical use. The aim of the present pre-clinical translation study was therefore to evaluate the effects of multiple low dose systemic injections of MNC hUCB cell into G93A SOD1 mice at different disease stages. Methodology/Principal Findings: Mice received weekly intravenous injections of MNC hUCB or media. Symptomatic mice received 10(6) or 2.5x10(6) cells from 13 weeks of age. A third, pre-symptomatic, group received 10(6) cells from 9 weeks of age. Control groups were media-injected G93A and mice carrying the normal hSOD1 gene. Motor function tests and various assays determined cell effects. Administered cell distribution, motor neuron counts, and glial cell densities were analyzed in mouse spinal cords. Results showed that mice receiving 10(6) cells pre-symptomatically or 2.5x10(6) cells symptomatically significantly delayed functional deterioration, increased lifespan and had higher motor neuron counts than media mice. Astrocytes and microglia were significantly reduced in all cell-treated groups. Conclusions/Significance: These results demonstrate that multiple injections of MNC hUCB cells, even beginning at the symptomatic disease stage, could benefit disease outcomes by protecting motor neurons from inflammatory effectors. This multiple cell infusion approach may promote future clinical studies.