Researcher: Alex Balduino de Souza/ Institute: UVA / IOC - RJ
New Method for Isolation of Multipotent Mesenchymal Stem Cells from Human Bone Marrow for Application in Cell Therapy Protocols.
The mesenchymal stem cells from bone marrow are defined by their ability to adhere to the substrate and the potential to differentiate into osteoblasts, chondrocytes and adipocytes. These features make these cells the main focus of study and development of future cell therapy protocols, particularly those applied to musculoskeletal tissues. Although widely used, the classical protocol for isolation / expansion of stem cells and mesenchymal progenitors from bone marrow, as well as in other tissues (fat, umbilical cord, etc.). Does not guarantee the purity or homogeneity of a population cells truly multipotent.
By clonogenic assays associated with assays of differentiation in vitro and in vivo, we know that the present method allows the isolation of a heterogeneous population of stem cells and committed progenitors bi- or unipotentes. This heterogeneity restricts its application in studies of regeneration of bone tissue, prevents the proper development of new protocols to differentiate into other cell types of musculoskeletal tissues (chondrocytes, osteoblasts and muscle cells) and hampers the development of clinical protocols for stem cell research expanded. Moreover, the use of a heterogeneous population of mesenchymal cells increases the risk of unwanted differentiation and proposals with clinical results inefficient and difficult to reproduce.
Although identified as distinct cells, mesenchymal stem cells and pericytes (perivascular cells) have broad similarity of the phenotype and differentiation potential of cells musculoskeletal tissues. Different authors describe that the isolated pericytes from skeletal striated muscle have adhesion late (only after 5 days) as compared with the myoblasts, which are the direct precursors of muscle fibers. Based on these data, our hypothesis is that the primary mesenchymal stem cells multipotent bone marrow, as well as pericytes present accession late compared with the other committed mesenchymal progenitors.
The new method proposed in the present design allows the insulation, expansion and characterization of the population of pluripotent cells from bone marrow. Furthermore, the purification of this population will be essential for studies of markers and mechanisms of differentiation of these cells, and serve mainly as a basis to broaden the spectrum of new cell therapies for other musculoskeletal tissues, and bone tissue. This new protocol will allow the differentiation potential of unorthodox cells (differentiation into neurons, glial cells, cardiomyocytes) is effective and properly evaluated.
Researcher: Alexander Henning Ulrich / Institute: USP
Molecular basis of differentiation of stem cells and neural progenitor in cell therapy.
The nervous system consists of a network composed of trillions of neurons with different phenotypes and other cell types of support. The wide variety of phenotypic cells in the nervous system is due to the differential development of stem cells and progenitor cells which give rise to neurons, astrocytes and oligodendrocytes. This process of neurogenesis is driven by activation of cell receptors on differentiation.
The participation of receptors for neurotransmitters and neuropeptides, such as the kinin B2 receptors, cholinergic and purinergic was studied and characterized by this laboratory during the neuronal differentiation in the model P19 embryonal carcinoma cells. Based on the obtained data, the study suggest the involvement of these receptors in neuronal differentiation, embryonic stem cells, mesenchymal cells from human adipose tissue and rat and the maturation of neurons functional from neural progenitor cells from mouse (neurospheres). To this end,will be developed appropriate DNA aptamers using the technique of Systematic evolution of ligands by exponential enrichment (SELEX) from a combinatorial library of DNA capable of specific binding to stem cells. Subsequently, these molecules will be used to purify these cells from adipose tissue.
The second stage of the project involves the application of mesenchymal stem cells and neural progenitor purified in vivo from peripheral sciatic nerve injury of rat in order to elucidate the therapeutic potential of these models. Furthermore, we will evaluate the ability of stem cells and progenitor yielding regenerative processes in the peripheral nervous system.
Researcher: Alfredo Miranda de Góes/ Institute: UFMG
Development of Constructs for Tissue Engineering by Stem Cell Cultivation of Human from Adipose Tissue in Three-Dimensional Structure of Biomaterials.
This project is focused on developing strategies for bone tissue engineering based on in vitro culture of stem cells from human adipose tissue previously grown on scaffolds three-dimensional. Relevant works have shown that stem cells from the supply of liposuction are able to differentiate into chondrogenic lineage, osteogenic, adipogenic and myogenic suggesting its applicability in tissue repair. The strategy used for engineering tissue in this design allows the cells to adhere to the support, proliferate and secrete specific extracellular matrix of bone tissue, to obtain an artificial substitute with functional characteristics of native tissue, which can ultimately be transplanted to treat the defect.
Therefore, the main objectives of this project are: (i) development of biodegradable scaffolds from new natural polymers based on cane sugar and chitosan to induce adhesion and cell proliferation and that exhibit suitable properties such as porosity and interconnectivity, to provide an environment that promotes in vitro development of a hybrid material with characteristics similar to human bone, (ii) analyzing the use of stem cells from human fat tissue as an alternative source of cells for engineering bone tissue, (iii) determine differentiation, survival and proliferation of stem cells from human adipose tissue in three-dimensional scaffolds, (iv) to evaluate the differential gene expression of stem cells after differentiation into osteoblasts on biomaterials using the proposed rash technique; (v) evaluate the profile of proinflammatory cytokines and anti-inflammatory receptor by the technique of real-time PCR and ELISA on the study after the allograft, (vi) evaluate the process of bone healing in ovariectomized rats (osteoporosis) after implantation of the biomaterial of stem cells.
These goals converge to the overall goal of the project is to develop a therapy for bone tissue engineering, alternative to existing, with the potential to be subsequently used in clinical practice and requiring a lower cost and patient rehabilitation.
Researcher: Ana Maria Blanco Martinez / Institute: UFRJ
Restorative Neuroscience: Strategies for Cell Transplantation in Animal Models of Sciatic Nerve Injury and Median and Spinal Cord in Mice.
Despite advances in surgical protocols and general care dispensed to patients with spinal cord injury, we do not have effective therapies for use in human clinical (Thuret et al., 2006, Andres et al., 2008; Eftekharpour et al., 2008). In recent decades there has been a breakthrough in the quest for cell therapies that restore functions lost by these patients. Stem cells from different origins, appear as potential sources of recovery of lost cells such as neurons and oligodendrocytes, which can restore lost contacts, build new circuits and / or myelinate spared axons, leading to significant functional improvement (Bradbury and McMahan, 2006 ; Cizkova et al., 2006, Li et al., 2007; Li and Raisman, 200).
In addition, these cells can release trophic factors, alternatively, may (a) to combat the inhibitory effects of extracellular matrix, particularly in areas of astrogliosis, (b) neutralizing the anti-regenerative substances released by oligodendrocytes, (c) inducing neural differentiation of implanted cells in vivo, (d) promote the differentiation of neural progenitor cells of the host, among other actions. These cells can be obtained from blastocysts, fetal and adult tissues of diverse lineages. Pre-clinical experimental studies have shown promising results in treating traumatic injuries of the spinal cord and peripheral nerves.
However, there is a great diversity of experimental models of injury, treatment strategies, cell lines and treatment of these cells, which hampers any conclusion in terms of the best protocol to be used in clinical medicine. Our experimental design is to use a single model of traumatic spinal cord injury, established in our laboratory (Marques et al., 2010), associated with treatments alone or in combination, through the use of different stem cells.
For treatment of injuries of peripheral nerves will use the model previously used by our group, where stem cells are grafted on nerves sectioned and using a tubular nerve guide. Our results with the latter strategy generated in grafted animals, a significant improvement, both from a functional standpoint as citoarquitetônico. These findings were published in Experimental Neurology in 2006.
We hope to continue contributing to this research area, with results easily reproducible, which can be translated into effective therapies for patients with traumatic injuries affecting the nervous system. Experience in surgical techniques, morphological and functional features of the project participants, combined with partnerships with laboratories led by researchers who manipulate stem cells, favor the development of the proposed project.
Researcher: Andrea Gonçalves Trentin / Institute: UFSC
Evaluation of biotechnology and therapeutic potential of stem cells isolated from human hair follicle.
Populations of epidermal stem cells have been found in specific sites of mammalian skin as the hair follicle where they reside in a discrete microenvironment known as the hair follicle bulge. Stem cells isolated hair follicle are multipotent, which can originate neurons, glial cells, keratinocytes, melanocytes and smooth muscle, and have the potential to regenerate the epidermis, hair follicles and sebaceous glands. Also, have properties similar to stem cells from embryonic neural crest. In our laboratory, cell culture conditions of the bulge of the hair follicle of mice were standardized, in which we identified, by RT-PCR and immunohistochemistry, the markers of stem cells from neural crest like p75, as well as neuronal marker nestin, glial and myofibroblasts. The hair follicle represents excellent model for further studies of stem cell niches and studies to identify the conditions that allow cells to adopt and maintain stem cell characteristics of long-lived and thus enable the development of protocols for regeneration and repair skin in addition to treatment of lesions of the nervous system, demyelinating diseases, vascular diseases, degenerative cartilage-bone, among others.
The burn has been identified as a major public health problem in many developing countries. The treatment of burns has evolved significantly in recent years with a better understanding of the pathophysiology in the acute phase and fluid replacement extensively burned patients, modulation of immune and inflammatory response. Several studies have been aimed at obtaining a integral skin, composed of dermis and epidermis, to be used in the tangential excisions of skin coverage. Models of dermic regeneration and of cell culture have been developed experimentally in clinical trials, however not yet been developed an optimal coverage. Thus, there is need to develop new therapeutic procedures. To this end, understanding the cellular and molecular mechanisms that control differentiation and self-renewal of epidermal stem cells are crucial.
Thus, in this project we intend to evaluate the therapeutic potential and biotechnological of stem cells from human hair follicles. Expected to characterize the human hair follicle as accessible and autologous source of stem cells, identifying conditions of differentiation and renewal of these cells. The project also provides an approach to the pre-clinical development of new coating skin which allows the regeneration of the dermal and epidermal layers in an animal model of mice. Thus we generate knowledge for the development of therapeutic and biotechnological processes.
Researcher: Anna Carla Renata Krepel Goldberg / Institute: USP
Differentiation of hepatocytes from stem cells from umbilical cord and development of preclinical test aimed transplantation in patients with hepatic impairment.
The significant morbidity and mortality in the list of patients waiting for liver transplantation, the only effective treatment for patients with liver failure, is a worldwide problem due to the shortage of donor organs. Faced with this problem, major research centers around the world seeking the development of therapies that can give temporary support to the metabolic functions of the liver, until an organ becomes available. The transplantation of hepatocytes, which is the transfer of a suspension of hepatocytes to a diseased liver, presented as a less invasive alternative to the transplantation of liver, may become a routine procedure to be used in situations acute liver failure, reducing or eliminating the clinical symptoms. There are reports of success in some cases treated with transplantation isolated human hepatocytes, however, this therapy has limited application due to the difficulty of obtaining such cells.
Based on these, it is important to look for alternative sources of isolated hepatocytes. In order to make the transplantation of hepatocytes a routine procedure to improve the quality of life of patients awaiting a liver transplant, this project aims at the differentiation of hepatocytes from human mesenchymal stem cells from umbilical cord and development of animal model of hepatic transplantation for validation of the transplantation differentiated hepatocytes.
The process of differentiation will be monitored by in vitro functional test using the techniques of real time PCR, for assessment of specific markers of liver, optical microscopy to assess the ability to produce albumin and storing glycogen, dosage in medium culture, alanine transaminase, total bilirubin, urea and albumin. These tests will be made in human hepatoma HepG2 cells (positive control) in mesenchymal stem cells did not undergo the differentiation protocol (negative control) and mesenchymal stem cells undergoing differentiation protocol. In parallel to differentiation protocols will be developed murine models of acute liver failure (70% hepatectomy) and chronic (cirrhosis induced by dimethylnitrosamine) for validation of the transplantation of hepatocytes. In the acute model, will be made three experimental groups: HepG2 cell transplantation, hepatocytes differentiated and simulation procedure (sham). In the chronic model, in addition, mesenchymal stem cells differentiated not also be deployed to evaluate the in vivo differentiation.
The metabolic support given by the transplanted cells to rats with liver failure, will be analyzed by serum and liver histological sections, using antibodies that discriminate against the production of human albumin albumin of the animal. The implementation of this method opens the way for setting not only the routine transplant bridge, but also the development of microencapsulation and cryopreserved hepatocytes for transplantation itself and improving the differentiation process by Tissue Engineering, based on studies already in development in NUCEL.
Researcher: Antonio Carlos Campos de Carvalho / Institute: UFRJ
Stem cells: therapeutic potential in heart disease.
At the turn of the Millenium, Regenerative Medicine has become an alternative for chronic degenerative diseases. In cardiac diseases, due to the high morbi-mortality, there is high interest for new therapeutic modalities. In this scenario cell therapy with different stem cell types has been intensively investigated. Our group has been working with basic, pre-clinic and clinical aspects of cell therapy. In basic research we are investigating the molecular mechanisms involved in cellular reprogramming and are searching for robust methods for differentiating pluripotent cells into cardiomyocytes, besides attempting to isolate and characterize distinct stem/progenitor cardiac stem cells. In pre-clinic research we are testing the effects of MSC from different sources and of cardiac stem cells in murine models of cardiac diseases, such as Chagas and ischemic heart diseases, evaluating action mechanisms and structural and functional benefits. In the clinical arena we have been testing bone-marrow derived cells in safety and efficacy trials in four cardiopathies: chagasic, idiopathic dilated, chronic and acute coronary heart disease. In addition we are generating iPSC from patients with cardiac diseases, focusing in cardiac arrhythmias.
Researcher: Beatriz Monteiro Long / Institute: UNIFESP
Evaluation of the neural precursor cell transplantation in experimental models of epilepsy.
Epilepsy's main characteristic is manifestation of spontaneous recurrent seizures that develop from neural changes in certain areas of the central nervous system (Dichter, 1997). Continuous and high-frequency neuronal firing can be generated by a surrounding inhibitory synaptic dysfunction. The crisis spread beyond the original focus can also be caused by an imbalance between excitation and inhibition. Therefore, the consequences of the loss or dysfunction of GABAergic interneurons in the development of the epilepticus framework are important. The imbalance of GABAergic neurotransmission, in addition to contributing to epileptogenesis, also has an important role in anxiety disorders that arise as a co-morbidity in epilepsy (Vazquez and Devinsky, 2003; Mendius and Perini, 1984, Altshuler et al, 1990).
Recent findings show that transplantation of neural precursor cells, derived from the medial ganglionic eminence (MGE), have an ability to migrate and differentiate into inhibitory interneurons in the cortical parenchyma of the host (Alvarez-Dolado et al., 2006). Therefore this project aims to evaluate the effect of transplantation of precursor interneurons in epileptogenic processes and behaviors associated with it.
Researcher: Bruno Dallagiovanna Muñiz / Institute: FIOCRUZ - RJ
Mechanisms of post-transcriptional regulation of self-renewal in human mesenchymal stem cells - Functional analysis of proteins and pumilio2 dzip1.
The target of cell transplantation is repopulate the transplant tissue or organ a patient with a new pool of cells that could restore the lost function. The mesenchymal stem cells (MSCs) are multipotent adult stem cells and has emerged as an option for use in regenerative medicine. The availability of MSCs were acquired from certain tissue donors, ease of expansion in culture, simple isolation via plastic adherence, the low rate of rejection and their multipotentiality for differentiation of MSCs makes it ideal for clinical applications. Recent studies indicate that the in vitro expansion of these cells is limited. MSCs senescem and lose their differentiation potential with increasing passage of time and culture in a process called "aging in vitro", which is an important limitation to their use in cell therapy.
A functional property of all types central stem cell is its ability to self-renewal, in which the stem cell is divided symmetrically to produce daughter cells with different destinations. Factors intracellular and extracellular control mechanisms that drive the daughter cell to self-renew or to commission a differentiation. The mechanisms that regulate gene level in the maintenance of self-renewal are not well known. The few existing studies focus on the analysis of the mechanisms regulating the initiation of transcription. However, the results observed both analyzing genes individually and transcriptome patterns highlight the importance of post-transcriptional regulation of genes involved in self-renewal.
The mechanisms that regulate post-transcriptional proteins are mediated by bonding to RNA, which preferentially bind to cis elements present in the untranslated regions of the mRNAs. Thus regulating the average life of the transcripts and which of them will finally be translated into protein. The networks formed by proteins and protein Pumilio2 DZIP1 are involved in self-renewal of embryonic stem cells and germ of many organisms, including humans suggesting their participation in the mechanisms of self-renewal of MSCs. The mechanisms that regulate gene level in the maintenance of self-renewal are not well known.
In this context, we intend to analyze the molecular mechanisms involved in the process of self-renewal and differentiation of MSCs in post-transcriptional level and the involvement of proteins PUM2 and DZIP1. By RT-PCR, we observed the expression of mRNA PUM2 and DZIP1 in MSC derived from bone marrow, umbilical cord blood and adipose tissue. We intend to conduct tests during the time course of differentiation of MSCs originating from adipose tissue during adipogenesis determining the expression of transcripts and proteins DZIP1 and PUM2. The pattern of expression during the differentiation process will infer the biological process they are involved. Testing by FISH localization of mRNAs reported that the changes observed are a general phenomenon restricted to a subpopulation of MSCs in differentiation. The cellular localization of the proteins studied depends on the activation state (differentiation) of stem cells. To understand the initial state of MSCs will carry out tests immunofluorescence of DZIP1 and PUM2 in MSCs.
In order to understand which signaling or metabolic pathways are regulated by proteins under study, it is essential to identify the mRNA targets. Immunoprecipitation assays will carry out the ribonucleoprotein complexes formed by DZIP1 and PUM2, and after purification of the associated, identify mRNAs by hybridization in human DNA microarray. Previously, the adjustment of technical conditions of this test will done on HeLa cells. Identifying the target mRNAs and proteins PUM2 and DZIP1 we characterize the genetic networks and concomitant metabolic pathways essential for the maintenance of the primitive state. These results are confirmed by EMSA and characterize the kinetic constants of interaction between proteins and transcripts regulated by them.
Finally the importance of the proteins studied in the maintenance of mechanisms to maintain the original state will be analyzed by performing tests of "knock down" gene using the technique of RNA interference. We will use commercial lentiviral vectors expressing small RNAs complementary to sequences of both genes. With a negative phenotype, we will analyze the differentiation potential of cells and expression of previously identified target mRNAs.
Knowledge of regulatory networks will allow to identify factors and signs inducers and repressors of the mechanisms of cell differentiation. In addition to providing important data for understanding fundamental biological processes that regulate the primitive state of MSCs, we can try to find alternatives to avoid the adverse effects of senescence and loss of in vitro differentiation capacity of mesenchymal stem cells.
Researcher: Carlos Alexandre Netto / Institute: UFRGS - RS
Use of human mesenchymal stem cells from umbilical cord blood and amniotic fluid in the functional regeneration of the spinal cord injury and cerebral hypoxia-ischemia in neonatal rats.
Regenerative medicine is the great innovation of this century. The study of stem cells (ES) is breaking paradigms, proving to be the future of regenerative therapy of organs and tissues. This research project classifies itself as pre-clinical research where mesenchymal stem cells from two different sources are used in animal models of spinal cord injury and neonatal cerebral hypoxia-ischemia. Cells of the umbilical cord and amniotic fluid will be grown to obtain the mesenchymal stem cells, which have been considered pluripotent, ie capable of generating various body tissues.
These cells will be isolated, cultured and characterized by the criteria established by the literature to be described them as MSC, which are: proliferation as adherent cells., immunophenotype and differentiation capacity in vitro. The molecular mechanisms of maintenance of the primitive state of ES will be evaluated through the analysis by RT-PCR for Oct4, a marker of the embryonic state. In addition, karyotyping is performed in cultured cells and cellular mechanisms are analyzed by flow cytometry. His induction of cell differentiation will be evaluated by differentiating into osteoblasts and adipocytes. Thus, well-characterized stem cells will be used in both animal models of neurological damage, which will study the application window after injury and route of administration of cells, as well as the morphological evaluation, functional, motor and cognitive.
Researcher: Cristina Ribeiro de Barros Cardoso / Institute: UFTM - MG
Therapeutic potential of mesenchymal stromal cells in inflammatory bowel disease.
Inflammatory Bowel Disease (IBD) such as Crohn's disease and ulcerative colitis, are characterized by chronic inflammation of the intestinal mucosa uncontrolled. As with ulcerative colitis is characterized by Th2 responses, patients with Crohn's disease show a Th1 and recently discovered that the presence of Th17 cells, with production of IL-17 and IL-23 in inflamed mucosa. Moreover, the regulatory T cell subsets have a critical role in controlling autoimmune responses and / or excessive, as in IBD. Although current therapies are directed to those elements of the immuno-inflammatory cascade, no treatment is at present fully effective, it is evident the need for new therapies to control the progression of IBD.
The mesenchymal stromal cells (MSCs) are multipotent cells from bone marrow, immunosuppressive and large capacity regulator, with significant therapeutic prospects for treatment of autoimmune and inflammatory diseases. Thus, this preclinical study aims to evaluate the therapeutic potential of human MSCs in experimentally induced IBD. The MSCs are isolated and cultured from bone marrow mononuclear cells from healthy donors. IBD is induced in Balb / c mice injected intra-rectal trinitrobenzene sulfonic acid (TNBS) and after induction of the disease phenotype and marking of MSCs, the animals are treated cells and sacrificed on days 7, 14 and 60 post-transplant for the collection of intestinal segments and mesenteric lymph nodes. Are evaluated weight, presence of diarrhea and, histologically, intestinal inflammatory response of mice with IBD before and after treatment with MSC. Modulation of intestinal immune response will be evaluated with emphasis on subpopulations of regulatory T cells and Th17.
Besides these, the inflammatory cells (CD3, CD4, CD8, CD11b, CD11c, NK, NKT, gamma-delta) in the intestine and lymph nodes are assessed by flow cytometry, immunohistochemistry and confocal microscopy. The expression of transcription factors ROR, Foxp3 and IL-10, TGF-ß, IL-4, IFN-gamma, TNF-alpha, IL-6, IL-17, IL-21, IL-23, IL- 25 and IL-27 will be assessed by real-time PCR and / or ELISA. Moreover, the potential suppression of regulatory T cells before and after treatment with MSCs be assessed by co-cultivation assays with effector cells as well as the proliferative capacity of effector T lymphocytes from animals with IBD front of MSCs.
The presence, in the intestine, the population of infused MSC, will be assessed by immunohistochemistry / confocal and the occurrence of cell fusion between MSC murine and human intestinal cells by FISH. Samples of other organs are also collected in order to verify the possible changes and migrating to other MSCs than those inflamed tissues, through the marking of the MSC (Qtracker) prior to infusion. Finally, the results obtained will help in understanding the pathogenesis of IBD and to demonstrate the effectiveness of MSC in the treatment of these diseases, as well as the mechanisms involved. Thus, this design aims at providing a scientific basis for the development of new therapeutic strategies for the treatment of ulcerative colitis and / or Crohn's disease.
Researcher: Dimas Tadeu Covas / Institute: USP - SP
Generation of induced pluripotent stem cells (iPS) using human adenoviral and lentiviral vectors.
Lines of embryonic stem cells (ESCs) derived from human inner cell mass of blastocysts are pluripotent and, besides having a great therapeutic potential as a source of tissues for transplantation are an important tool for basic research focused on the study of cell differentiation and development human embryonic, among others. Although in animal models been demonstrated the therapeutic effect of ESCs in various diseases, the clinical use of these cells in human-beings has not been reported. Issues related to immunocompatibility between tissues derived from ESCs and the patient, among others, remain to be resolved before they can be used in clinical trials.
Recently two groups have reported the induction of pluripotency in primary human fibroblasts through the same transduction with viral vectors expressing sets of four different transcription factors (TF): OCT4, NANOG, LIM28 and SOX2, or OCT4, C-MYC and KLF4 SOX2. The so-called pluripotent stem cells induced (iPS) have a characteristic morphology of ESCs, pluripotent cells express markers and are able to differentiate in vitro and in vivo in tissues derived from the three germ layers. Although still inadequate for clinical use, the iPS are an important tool for basic research, especially those cells derived from individuals with different genetic diseases.
In this project, we intend to develop new reprogramming vectors (lentiviral and adenoviral) to be used in the reprogramming of mesenchymal stem cells (MSC) and endothelial progenitor cells. MSC and endothelial progenitors derived from human bone marrow are considered the most promising stem cells for cell therapy because of its ability to expand in vitro and their potential of differentiation. However, these cells are limited to differentiate into cells of mesodermal origin and only after successive passages become senescent. The addition of TF related to pluripotency can turn these into pluripotent and multipotent cells capable of unlimited expansion in vitro.
In the first phase of the project will use lentiviral vectors, due to the extensive experience of the group with these vectors, to standardize the tests transduction, analyze the effects of TF on mesenchymal stem cells and in endothelial progenitor and determine which group of TF are required to generate iPS lines of these cells. The second phase will be chosen TF indispensable for iPS generation that will be cloned into adenoviral vectors to generate iPS not genetically modified to be used in clinical trials.
Researcher: Emerson Leandro Gasparetto / Institute: UFRJ - RJ
Tagging stem cells with superparamagnetic nanoparticles to assess the migration and integration of these in animal models of cell therapies.
Stem cells have been used in several animal models of diseases and injuries of the central nervous system and also with promising results in clinical studies. One of the problems in these therapies is the difficulty in assessing the migration of these cells to areas of injury as well as check their possible integration with the host tissue and its possible differentiation. This project will evaluate the ability to mark pluripotent stem cells (embryonic, neural and induced) and adult stem cells (mononuclear and mesenchymal cells derived from bone marrow and umbilical cord blood) with superparamagnetic nanoparticles of iron oxide (Superparamagnetic Iron Oxide Nanoparticles - Spion). This marking allows the dynamic monitoring of cells within the recipient organism by nuclear magnetic resonance (MR) imaging technique available today in hospitals and medical research. Although the magnetic properties of iron ions are essential for the observation by MRI is also necessary to take these nanoparticles with substances that facilitate cell permeation and absorption of these nanoparticles by target cells. An example is coated with dextran, a polysaccharide, and these coated particles now being routinely used in MRI scans, and available in different trade names such as, for example, Feridex®. The particles are available commercially, however, are designed to mark phagocytic cells since they are rapidly taken up by macrophages, monocytes and Kupfer cells for example. Stem cells but are not phagocytic and protocols marking must be developed to promote marking cells with these nanoparticles without altering the biological and therapeutic properties thereof.
Our proposal is to prepare SPIONs coated with different materials to allow a greater permeability through the cell membrane, such as protamine and other cationic polypeptides. Furthermore, we want to develop SPIONs coated with a monolayer of silica support in order to permit determination of ligands specific to these receptor proteins in cell lines established. This methodology will provide a selectivity in the labeling of subpopulations of cells for cell therapy, for example, the population of precursor endotelias within the mononuclear fraction and either sub-population of mesenchymal cells. With this methodology may mark specific populations for therapies for myocardial infarction or stroke, or to map the presence of cholinergic neurons in the CNS and follow the evolution of his disappearance in neurodegenerative diseases like Alzheimer's. These labeled cells are injected into different models of neurological diseases and migration and integration of labeled cells in the areas of tissue degeneration will be evaluated by histochemical methods, immunohistochemical and image. Once established the protocols for tagging these stem cells could be used by different groups working on cell therapies in various animal models. Once the safety and efficacy of these labeled cells is demonstrated in animal models will be possible to extend its use for clinical trials of cell therapies.
Researcher: Geraldo A. Passos / Institute: USP / Ribeirão Preto - SP
_Comparative analysis of the transcriptional expression of syntenic genes of murine and human mesenchymal stem cells.
Recent studies indicate that mesenchymal cells from specific tissues are able to differentiate into cells of other tissues, thus offering the prospect of new therapeutic strategies for the repair of damaged tissues. The therapeutic potential of stem cells is already a reality, but many aspects of molecular biology of proliferation and differentiation of these cells remain unknown. We do not know all the genes involved in the process of differentiation of these cells. To achieve this knowledge we must use tools of functional genomics and measured in terms the differential expression of genes on a large scale during differentiation, thus performing the analysis of the transcriptome.
The technology of DNA microarrays is consecrated in the area of gene expression analysis on a large scale, widely applied in biology and medicine in the acquisition of new basic knowledge and also in the diagnostic field. With DNA microarrays and applying mathematical-statistical methods specific, we can measure the expression of thousands of genes in a single experiment. Therefore microarrays have become the method of choice in the exploration of the transcriptome in various normal and pathological situations.
Genomic comparisons between species is very important. Synteny is the term used to conceptualize the inter-species conservation of gene order along the chromosomes and whose sequence homology between these genes also matters. Studies on the human-mouse synteny represent great interest, since they contribute to research on the coincidence of the molecular genetic events that occur in human cells and in murine model system. Since there is obvious difficulty in obtaining stem cells from human bone marrow mesenchymal cells for basic research, the murine model system becomes of great interest.
It is noteworthy that comparative studies on osteogenesis, markers of osteoblastic phenotype and gene expression profiling using DNA microarrays for stem cells, bone marrow derived murine and human bone marrow, before and after they were differentiated into osteoblasts are unique, as well as analysis the syntenic gene expression profiling are still required. There are no data in the literature on this type of comparison is important to consider and accomplish these studies, because we can establish the genetic and molecular parameters that can be best studied using murine instead of human for obvious reasons.
Researcher: Helena Paula Brentani / Institute: FAP-SP
Comparative analysis of the mechanisms of neurogenesis of cells induced pluripotent stem (iPS) generated from keratinocytes from normal and schizophrenic patients.
Schizophrenia is a mental illness that causes changes in the process of thoughts, perceptions and emotions and normally leads to a deterioration and affective blunting. Adoption studies, twin, association and linkage have shown that there is a strong genetic component in schizophrenia. The heterogeneity and complexity of the tables has challenged phenotypic linkage studies. Recent studies have brought replicated evidence that patients suffering from schizophrenia have increased structural variations along its genome. So we can categorize schizophrenia, and autism and other mental retardation, such as genomic disorders of neurodevelopment. Cells induced pluripotent stem (iPS) are obtained by reprogramming adult skin cells. Because they are genetically identical to the donor, and capable of generating all cell types including neurons of an individual, may aid in the characterization of alterations in the development of the nervous system associated with specific diseases and difficult etiology of schizophrenia.
This project is proposed to study possible changes in gene expression and phenotypic characteristics of neurons produced from iPS generated by reprogramming keratinocytes from schizophrenic patients with a family history well-characterized compared with iPS from normal subjects.
The proposal is innovative, unique and has applicability and relevance to the scientific , technological and clinical development of our country.
Researcher: Ilka Boin / Institute: Unicamp - SP
Mesenchymal stem cells obtained from human adipocyte tissue umbilical cord blood and bone marrow and their ability to differentiate into functional hepatocytes.
Liver cirrhosis results in loss of cellular mass and great diminish of liver function. Stem cell therapy could be a possible alternative to liver transplantation. The aim of this study is to analyze if mesenchymal stem cells (MSCs) obtained from human adipocyte tissue (AT), umbilical cord blood (UBC) and bone marrow could differentiate into functional hepatocytes. Functionality will be analyzed through their transplantation into SCID mouse submitted to fulminant hepatitis secondary to intraperitoneal injection of carbon tetrachlorid (CCL4).
Material and Method: AT will be obtained from lipoaspirate procedures, performed in the Clinic Hospital, UCB from units collected by the Public Cord Blood Bank and bone marrow from units donated to Bone Marrow Transplantation Unit, ,all the services from State University Of Campinas. AT will be submitted to collagenase digestion, UCB and BM to Ficoll gradient. Cells will be cultured in DMEM low glucose medium, with FBS during 3 days. At 80% of confluence, adherent cells are treated with trypsin and harvested with the medium above, first passage. At the fourth passage, cells are characterized by flow cytometry, confocal microscopy, differentiated to mesodermal lineages to confirm the differentiation to MSCs. Telomerase enzyme activity and karyotype analysis will also be performed. MSCs will be submitted to hepatocyte differentiation by a three steps culture. Step 0;MSCs cultured with DMEM low glucose, 20ng/mL of EGF, 10 ng/ mL of bFGF, serum deprived for two days. Step 1: DMEM low glucose, 15% FBS, 20ng/mL HGF, 10ng/mL bFGF, 0.61g/L of nicotinamide for 7 days Step 3: Addition of maturation medium (20ng/mL of oncostatin, 1µmol/L of dexamethasone and 50mg/mL of ITS), for 36 days. Hepatogenesis will be analyzed by morphology (light microscopy), functionality (detection of glycogen storage, (PAS stain), and indocyanine green, (ICG, absorption), and gene expression by RT-PCR of albumin (A), Alpha- fetoprotein (AF), and tyrosine amino transferase( TAT) and glutamine synthetase (GS) genes on day 9, 18, 25 and 36. Animal experiments will be performed with male Balbc/SCID mouse with 6- 8 weeks of age the AT, UCB and BM donors were female. They will be submitted to fulminant hepatitis by intraperitoneal injection of 500 µg/kg diluted in 100 uL of virgin oil. Pre–labeled undifferentiated and differentiated MSCs of the three sources will be injected into tail or orbital vein 48 hrs post CCL4 injection. The CCL4 treatment efficacy will be demonstrated by the anatomo-pathological results. The animals will be sacrificed 24hrs, 48hrs, 7 0, 150 and 300 days post CCL4 injection, After transplantation the animals will be sacrificed on the 7 0, 150 and 300 days. The transplantation results will be performed by anatomo-pathological analysis, light microscopy (HE for morphological analysis and Masson dye for fibrosis detection). Homing and “in situ” proliferation of pre–labeled (fluorescent dye) undifferentiated and differentiated MSCs of the three sources in injured liver will be analyzed by fluorescent microscope and Fish test. Functionality will be held by hepatic enzymes AST and ALT analysis pre and post CCL4 injection and post transplantation ON THE 7 0, 150 and 300 days. Albumin secretion will also be performed during the periods analyzed.
Researcher: Jose Eduardo Krieger / Institute: USP-SP
Reprogramming adult cells of adipose tissue in pluripotent stem cells and cardiomyocytes.
Reprogramming successful human somatic cells a state of pluripotency allow the creation of stem cell disease and patient specific. Recently it was shown that by introducing at least four transcription factors (Sox2, Oct3 / 4, KLF4 and c-Myc) by viral transduction in somatic cells, these are reprogrammed to a pluripotent state very similar to the ES. These cells are called induced pluripotent stem (iPS). In subsequent work, the same group led by Yamanaka was able to induce the differentiation of these cells into cardiomyocytes, and observed improvement in cardiac function when injected into infarction model. However the efficiency of this differentiation process is very low, making it difficult to use in humans. Furthermore, the use of retroviral vectors makes this procedure reduced the clinical application because of the risks of development of tumors by endogenous gene activation caused by insertional mutagenesis.
To circumvent these problems, this paper we want: 1. iPS establish cell culture from mice and human fibroblasts according to the technique developed by Takahashi and Yamanaka; 2. test the hypothesis that iPS cells can become cardiomyocytes efficiently through the transduction of transcription factors that induce cardiomyogenesis; 3. test the hypothesis that the action of transcription factors linked to a secretion signal peptide and the TAT carrier reprogram mesenchymal cells of adipose tissue in cells induced pluripotent (iPS) that does not require the use of viruses in the process. Thus, we want to obtain specific cardiomyocytes more efficiently than could be used to repair cardiac biological cell therapy using cells from the patient.
Researcher: Joyce Maria Annichino-Bizzacchi / Institute: UNICAMP-SP
Evaluation of the use of human stem cells derived from adipose tissue in neovascularization of ischemic limbs of mice - preclinical study cell therapy in Peripheral Arterial Disease Chronic (PADC).
Peripheral arterial disease (PAD) is a chronic arterial occlusive disease of the lower limbs caused by atherosclerosis. It is one of the most common causes of pain and disability of the lower limbs due to the neglect of their diagnosis, the PADC is associated with significant morbidity and mortality. The angiogenic therapy using stem cells is an alternative treatment of the resulting cell plasticity promoting neovascularization. The objective of this work is to promote neovascularization in ischemic limbs of murine models of PADC, by the use of stem cells derived from adipose tissue, from normal human donors and evaluate their effectiveness for future cell therapy-based treatments. The use of stem cells derived from adipose tissue is a new promising market for obtaining adult stem cells. The aim is to validate an animal model that mimics human PADC properly, allowing a better assessment of response to cell therapy. This therapy will be evaluated by the combination of the methods of relative perfusion, capillary density, capillary permeability and arteriogenesis.
Researcher: Julio Cesar Voltarelli / Institute: USP-SP
Therapy of diabetes mellitus with mesenchymal stem cells: evaluation of clinical response and investigation of mechanisms of action.
The type 1 diabetes mellitus (DM-1) is an autoimmune disease (AID) inflammatory mediated by auto-reactive T cells and characterized by the selective destruction of pancreatic beta cells that produce insulin. The susceptibility to DM-1 is determined by genetic factors, environmental and defective mechanisms of immunoregulation. Clinical disease is manifested only after approximately 80-90% destruction of pancreatic B cells. The b-cell destructive process leads to a lack of the hormone insulin, which results in hyperglycemia and its acute and chronic consequences. If left untreated, metabolic disturbances lead progressively to central nervous system depression, coma and death. Thus, patients need permanent treatment with exogenous insulin for survival. Some serious complications arising from chronic autoimmune reaction are vascular problems leading to kidney failure, blindness, heart disease and chronic ulcers.
On the other hand, type 2 diabetes mellitus (DM-2) is a metabolic disorder associated primarily two mechanisms, the resistance to insulin action and secretion dysfunction pancreatic beta cells. The molecular mechanisms that determine the deficiency of insulin secretion or its deficient action are not well defined and are the subject of intense research in recent years. However, there is abundant evidence of the presence of an important inflammatory component involved in the pathogenesis of insulin resistance and pancreatic beta cell dysfunction. Insulin resistance, a major pathogenic factors of DM-2, is often associated with other cardiovascular risk factors such as hypertension, obesity and dyslipidemia. In the last decade, DM 2, which represents 90-95% of cases of diabetes has become a major problem in clinical and public health worldwide. The chronic complications of DM-2, which settle in the long run, result from micro-and macrovascular changes that lead to dysfunction, damage or failure of various organs. All this framework incurs high social and economic cost, maintaining productivity, quality of life and survival of the population.
The DM-1 is treated by conventional therapy with intensive therapy with insulin or insulin. Clinical trials with immunosuppressants, anti-CD3 monoclonal antibody and pancreatic islet transplantation have been conducted with significant results, but not sufficient for routine clinical application. Already, the treatment of DM-2 involves diet, exercise, oral drugs and often daily injections of insulin. This scenario encourages further research about alternative therapies for diabetes, such as in vitro differentiation of human stem cells in pancreatic β cells for transplantation and treatment of DM-1 with adult stem cells. In this context, mesenchymal stem cells (MSCs) represent a source of adult stem cells ideal for cell therapy because of its easy isolation, in vitro expansion and immunosuppressive capacity, regenerative and immunoregulatory.
Recent studies have demonstrated a satisfactory therapeutic effect after infusion of MSC in animal models of diabetes-1 (early in the disease) and in patients with chronic graft-versus-host disease whose immune mechanisms similar to those of AID. Additionally, there observations in experimental studies suggest that the beneficial effects of transplantation of stem cells from bone marrow total of hyperglycemia in diabetes and its complications-2. In this context, our hypothesis is that diabetes can be controlled through the infusion of mesenchymal stem cells, because once infused in diabetic patients they migrate preferentially to inflamed pancreatic tissue, promote local immunosuppression and stimulate the regeneration mechanisms through local paracrine effects, the which involve the production of anti-inflammatory molecules and imunoreguladoras and growth factors and angiogenic.
Considering the need for new therapies for the treatment of diabetes and the intrinsic properties of the MSC, we propose this project to evaluate the safety, therapeutic effect and mechanism of action of allogeneic MSC infusion in the treatment of DM-1 newly diagnosed in humans and in animal models of DM-1 and MD-2.
Evaluate the immune response (the frequency of naïve T-cell subpopulations, and memory T cell regulatory cytokine in serum T cell repertoire, frequency of self-reactive T cell anti-pancreatic beta cells) in patients at different times after infusion of MSC. In parallel to human studies, we propose this project to evaluate the mechanisms involved in immune and regenerative therapeutic response, the infusion of MSC, two models of autoimmune diabetes type 1 (a genetically determined model and the other chemically induced) in the initial phase and phase chronic disease. In addition, this project will evaluate the therapeutic effect of MSC infusion in mice with MD-2 as well as the mechanisms involved in immune and regenerative therapeutic response. In these animal models to evaluate DM-1 and MD-2 modulation of pathogenic autoimmune response MSC, as well as the role of MSC in the regeneration of pancreatic tissue injured by the autoimmune reaction.
The results of this project will demonstrate the therapeutic effect of MSC infusion in the treatment of DM-1 in humans newly diagnosed and chronic DM-1 and DM-2 in experimental models, as well as elucidate the mechanisms involved in immune and regenerative therapeutic response of diabetic animals to the infusion of MSC. If shown a good therapeutic efficacy without the toxic effects relevant, the infusion of MSC can become an alternative therapy for the treatment of DM-1 and DM-2, which are diseases associated with poor quality of life in long-term and chronic treatment of high cost.
Researcher: Manoel João Batista Castello Girão / Institute: UNIFESP-SP
Histomorphometric and immunohistochemical study of the urethra of adult rats injected with stem cells from bone marrow after simulated trauma of childbirth and in adult females with cell injection.
Urinary incontinence is a condition resulting in involuntary loss of urine. As for stress urinary incontinence is defined as any leakage of urine due to some physical exertion such as jumping, running and coughing. We emphasize the number of pregnancies and mode of delivery as predisposing factors, as they may damage the suspension system and support the pelvic organs.
Injury to the pelvic floor is, in general, by the compression of fetal parts from maternal tissues, which determines section and stretch muscles and nerves, and also structural disarrangement of connective tissue and fascia, changing all the static pelvic and may cause loss of urine.
Regenerative medicine is a new field of medicine that combines different fields of tissue engineering. Among these, there is the use of stem cells, which develop biological substitutes to restore and maintain the original functions of organs and tissues. There are several types of studies with cells of different origins for this purpose.
The objective of this study is to evaluate the possibility of regeneration of the pelvic floor, urethra and bladder for embryonic stem cells of female mice after simulated trauma of childbirth, as well as adult rats with adult stem cells from bone marrow. Tissues to be analyzed after birth trauma and cell therapy are muscle, nerve, vessel and collagen.
Researcher: Mari Cleide Sogayar / Institute: USP-SP
Differentiation of embryonic stem cells, mesenchymal skin cells and dental pulp cells into osteoblasts and pancreatic beta cells aiming cell therapy at degenerative diseases.
Diabetes mellitus (DM) is a major cause of morbidity and mortality in the world, can be classified as type 1 and 2. The DM1 is caused primarily by the destruction of pancreatic beta cells by autoimmune processes, characterized by absolute insulin deficiency. There is great difficulty in obtaining pancreas transplants, both total and organ islets. Thus, the differentiation of pancreatic beta cells from stem cell becomes an attractive possibility as an alternative for obtaining sufficient mass of β cells for transplantation. One promising source of beta cells are embryonic stem cells. The differentiation of murine embryonic stem cells into β cells, secreting insulin was successfully performed in our laboratory.
However, in spite of producing insulin, these cells were not effective to reverse diabetes in a preclinical model of diabetic mice, probably due to low viability of transplanted cells. This framework could be reversed through an innovative methodology recently implemented in our laboratory, which consists of microencapsulate islet cells or to increase their viability. Another source for β cells are adult stem cells, found in various tissues of adult individuals, including: bone marrow, neural tissue, retina, muscle, skin and teeth.
The isolation and cultivation of two strains of stem cells from human dental pulp and three strains of stem cells from murine skin was established in our laboratory. These tissues are to available sources of biological material to obtain cells. These cells showed a wide range of differentiation, which makes them good candidates for cell therapy of osteo-diferentiation. The trauma and bone fractures in the elderly represent a serious public health problem. One promising approach is given by treatment with BMPs, which in clinical trials and preclinical studies have shown to be effective in inducing ectopic bone formation in mice and humans, and reduce hospitalization time and costs, and also prevent a recurrence fracture. Recently, were produced in our laboratory, the recombinant human BMP2 and BMP7 (rhBMP2 and rhBMP7) in mammalian cells.
These proteins have been shown to induce osteogenesis in cellular model of pre-myoblasts (C2C12 mouse strain). The purification of these recombinant proteins and their use together with stem cells opens up promising perspectives since various pre-clinical studies demonstrated that induce regeneration of bone and cartilage tissue, further accelerating the recovery of inter-vertebral disc in procedures of arthrodesis. Thus, we propose an approach for differentiation of stem cells derived from easily obtainable tissue as the skin and dental pulp, into producing cells and insulin-secreting. We aim also to increase the survival time of these stem cells in vivo via its encapsulation. These same cells are used for osteo-diferentiation through exposure to rhBMP 2 and 7, in an attempt to generate protocols that are effective in the repair of damage or bone lesions.
Researcher: Maria Angelica Miglino / Institute: USP-SP
Use of human stem cells from immature tooth pulp in experimental animal models of acquired and traumatic neurological diseases.
In the last two decades, with the advancement of knowledge and molecular techniques, science was able to generate important information for the diagnosis and treatment of a series of previously untreated diseases. However, bone diseases and spinal cord injuries are still no effective treatment. Bone defects are responsible for the loss of work capacity and quality of life for millions of people around the world and constitute a public health problem, as found in traumatic spinal cord injury, where you can observe a high rate of patients, mostly young, predominantly male, in the peak of his productivity. The morbidity and mortality is high and its incidence increases about 4% per year. The involvement of bone and nervous system that occurs in these cases remains a medical challenge for the lack of effective tools in solving these problems and now, through investments in research in this area, some promising results are reported. Studies with animal models has shown that stem cell transplantation can be successful in treating many chronic diseases such osteogenic defects, spinal cord diseases, muscular dystrophy, Parkinson's, diabetes, degeneration of cells Purkinjie, liver and heart failure. Therefore we believe that are necessary studies that present the main objective of contribution in improving the quality of life of these patients, therefore, this study provide two animal models: 1. sheep, which aims to contribute to the bone defects, and; 2. canines, who have the spinal cord injury similar to humans.For the pre-clinical models, for bone diseases, for correcting it is necessary to use grafts that may be bony or biomaterials. However, the disadvantages and limitations of these methods available for such reconstructions makes the search for a less invasive, safer and more predictable, an immediate need. The bioengineered tissue, which works with the triad cells / scaffold / signaling molecules have been shown to be very promising for such purposes. In this study we will use as a scaffold bioceramic hydroxyapatite (HA) and ß-tricalcium phosphate (ß-TCP), in the form of granules and blocks, which are grown on isolated cells from dental pulp, called IDPSC (isolated and characterized by our group ) for obtaining a complex three-dimensional. The osteoblast differentiation occur through modifications of their culture media supplemented with differentiation factors. The clinical application will consist in the use of complex three-dimensional selected for the reconstruction of critical size bone defects performed in the tibia of sheep. Results will be evaluated histomorphometrically. For spinal cord injury in a canine model, with those already acquired will be treated with decompressive surgery followed by transplantation of cells from dental pulp, however a group receive the same cells through a puncture in the region of space atlanto-occipital. Our group isolated and characterized stem cells from immature tooth pulp (IDPSC). Due to its characteristics, these cells can be classified as multipotent adult stem cells (MSC), which is a cell type is interposed between the embryonic stem cells and adult stem cells. We also demonstrate that the present IDPSC expression profile is similar to that observed in human ES, without risk of rejection or teratoma formation. For this purpose, we use these types of stem cells in treatment of traumatic and acquired neurological injuries.
Researcher: Maria Isabel Doria Rossi / Institute: UFRJ, RJ
_Role of Cellular Interactions in Migration and Proliferation of Hematopoietic Progenitors and Tumor Cells, and in Osteogenic Differentiation of Mesenchymal Stromal Cells.
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Advances in tissue engineering and cell therapy raised scientific questions related to the cellular interactions that form an informative microenvironment that controls fate decisions of adult and cancer stem cells. The aim is to investigate mechanisms underlying differentiation of human mesenchymal stromal cells derived from bone marrow and their role in the organization of a three dimensional stem cell niche that allows migration of hematopoietic progenitors and neoplastic cells. Particular attention is given to the role of Wnt signaling pathway, adhesion molecules (cadherins and integrins) and extracellular matrix proteins (galectin-3 and proteoglycans) in: (i) osteogenic differentiation of bone marrow mesenchymal stromal cells; (ii) invasive behavior of breast cancer tumor cells with the phenotype of cancer stem cell; and (iii) epithelial to mesenchymal transition of breast cell lines. The differentiation potential of human mesenchymal stromal cells isolated from different sources will be investigated in embryonic microenvironment that allows interactions between the different germ layers.
Advances in tissue engineering and cell therapy raised scientific questions related to the cellular interactions that form an informative microenvironment that controls fate decisions of adult and cancer stem cells. The aim is to investigate mechanisms underlying differentiation of human mesenchymal stromal cells derived from bone marrow and their role in the organization of a three dimensional stem cell niche that allows migration of hematopoietic progenitors and neoplastic cells. Particular attention is given to the role of Wnt signaling pathway, adhesion molecules (cadherins and integrins) and extracellular matrix proteins (galectin-3 and proteoglycans) in: (i) osteogenic differentiation of bone marrow mesenchymal stromal cells; (ii) invasive behavior of breast cancer tumor cells with the phenotype of cancer stem cell; and (iii) epithelial to mesenchymal transition of breast cell lines. The differentiation potential of human mesenchymal stromal cells isolated from different sources will be investigated in embryonic microenvironment that allows interactions between the different germ layers.
Researcher: Maria Rita Passos Bueno / Institute: USP-SP
Stem Cells in Understanding and Rehabilitation of Craniofacial Disease.
Our research group is mainly focused on investing in stem cell research to improve the rehabilitation of patients with craniofacial syndromes, with less morbidity and postoperative complications. In the last three years, when we began research in this area, we characterize an animal model for the study of cranial bone reconstruction with stem cells attached to biomaterials, as well as feature a new source of stem cells from fragments of the orbicularis oris muscle, which are regularly discarded in corrective surgery of patients with cleft lip and palate (Costa et al., 2008, Bueno et al. 2007, Bueno et al., 2008). In this proposal, will continue to better characterize stem cells derived from the orbicular muscle of the lip, such as check whether they have the same potential osteogenic of tooth pulp cells, using the already established animal model.
In addition, we intend to: a) to identify genes that act during embryonic development and are critical for osteogenesis of craniofacial bones; to achieve this goal we will perform functional studies on stem cells obtained from patients with craniofacial syndromes, b) develop an animal model that is equivalent to that of cleft lip and palate and evaluate the potential of this defect bone regeneration with stem cells combined with biomaterials; c) determine whether stem cells combined with genes important for angiogenesis could lead to a more efficient ossification d) developing biomaterials national, combined with stem cells, are efficient in the process of craniofacial bone regeneration. For this project we organized a multidisciplinary team of professionals with expertise in the field of gene therapy (Professor Menck) and biomaterials (Professor Elson Longo).
Researcher: Marimélia Porcionatto / Institute: UNIFESP-SP
Effect of peptides derived chemokine SDF-1/CXCL12 on migration, proliferation, survival and differentiation of neural stem cells.
Physical trauma and stroke cause central nervous system lesions that represent a major health problem. Currently, there is no specific treatment for injuries such as trauma, stroke and spinal cord injuries. Of all the neurological diseases of adult life, stroke ranks first in frequency and importance, being the third leading cause of death and the leading cause of permanent disability in the West. Traumatic brain injury and spinal cord injury resulting from sudden and external physical harm to the head or spinal cord often have cognitive and motors deficits as a consequence with varying degrees of severity. The central nervous system damage resulting in glial reaction leading to the formation of a scar consisting in the deposition of extracellular matrix of inhibitory factors of remyelination and axonal extension. These molecules are secreted by reactive astrocytes and oligodendrocytes that migrate to the injury site.
The inability of regenerating nerve fibers leads to the death of the corresponding cell body and the undamaged fibers in regions close to the damaged axons are affected by lateral spreading of the injury and undergo secondary degeneration. Additional therapeutic strategies have been tested, such as allogeneic stem cells derived from bone marrow in spinal cord injury, but these treatments have not yet produced the desired effects. Presumably, intrinsic recovery of this type of injury are linked to the presence of endogenous neural stem cells that seem to be able to act as a neuroprotective and / or neuroregeneratives.
The endogenous stem cells present in neurogenic regions and the subventricular zone of the hippocampus and subgranular layer migrate to the injury in response to chemotactic stimuli secreted by glial cells and immune cells present in the lesion. One of the factors involved in stimulating chemotactic migration of stem cells is the endogenous cytokine SDF-1/CXCL12. Although it has been shown that neural stem cells are able to reach the lesion, many of them die by mechanisms still unknown. Possibly, the anti and pro-inflammatory cytokines present in the lesion, influencing not only in chemotaxis but also the final destination of neural stem cells that reach the lesion. In addition to cytokines and growth inhibitory molecules axonal, reactive glia increases the expression of matrix metalloproteases (MMPs) that, among other things, degrade certain cytokines. Already described in the literature that the degradation by MMPs SDF-1/CXCL12 cause inactivation of chemokine, however, it is possible that cleavage SDF-1/CXCL12 generate bioactive peptides that could act as stimulators of differentiation and death inhibitors cell. Thus, this project aims to study the migration of neural stem cells stimulated by peptides derived from SDF-1/CXCL12 in vitro, as well as evaluating the effects of these peptides on the proliferation, survival and differentiation of neural stem cells.
We hope that the results obtained in this project can clarify important points about the cellular and molecular mechanisms of chemotaxis, survival and differentiation of endogenous neural stem cells in response to injury in the central nervous system and to indicate possible future treatment by increased chemotactic activity for structural changes in synthetic peptides, as well as by chemical manipulation of the enzymes involved in processing SDF-1/CXCL12 in vivo.
Researcher: Mauro Martins Teixeira / Institute: UFMG - MG
Preclinical study of the therapeutic use of endothelial progenitor cells preconditioned ex vivo in the revascularization of ischemic tissues.
Peripheral arterial disease, also known as peripheral vascular disease is a chronic disease that progressively impairs arterial circulation of the limbs. In general, these arterial damage are secondary to hypertension and / or the formation of atherosclerotic plaques. If treatment with medication or surgical or endovascular procedures are not successful, most of these patients will suffer the amputation of the affected limbs. Consequently, the exploration of new therapeutic strategies for revascularization of ischemic tissues is of great importance.
In the last decade, have been researched therapies aimed at increasing blood flow to ischemic tissues by transplanting stem cells that promote local development of new blood vessels. For example, cell therapy based on transplantation of endothelial progenitor cells (EPC), could not only result in the differentiation of these cells to mature endothelial cells (vasculogenesis) and thus contribute to the reparation neovascularization, but also in the production of various growth factors can stimulate angiogenesis (new blood vessels from pre-existents vessels) and arteriogenesis (collateral vessel formation) through paracrine mechanisms.
Clinical studies have reported promising results of the EPC-based therapy for the treatment of chronic limb ischemia, however, the results are still modest when compared to pre-clinical studies. Recent discovery shows that bradykinin acting on B2 receptors in EPCs and through activating the Akt / eNOS is one of the mechanisms involved in the migration of these cells to ischemic tissues. Inhibitors of angiotensin I-converting enzyme (ACE), widely used as antihypertensive agents, were shown to possess pro-angiogenic effects mediated by activation of bradykinin B2 receptor by blockade mechanisms independent of inactivation of the enzyme. Similar mechanisms are also observed in the antihypertensive effects of angiotensin-(1-7). Interestingly, the potentiating effect of bradykinin as a result of treatment with ACE inhibitors can be reversed by a MAS receptor antagonist of angiotensin-(1-7). The enhancer activity of bradykinin (BK) is the best known biological property of peptides isolated from the venom of Bothrops jararaca (BPPs, the acronym "bradykinin-potentiating peptide"). To this was initially attributed to an inhibitory effect on ACE.
However, recently it was shown that the antihypertensive activities of BPPs can also occur through independent mechanisms of ACE, such as, for example, the increased production of nitric oxide by direct binding to the enzyme argininosuccinate synthase (AsS). The pharmacological enhancement of adhesive and migratory functions of the EPC, the ability to incorporate these blood vessels in training or even the ability to release angiogenic factors represents a new area of research that may lead to the development of new strategies for increasing the efficiency transplantation of EPCs. Since BPPs are able to activate functions of angiogenic endothelial cells (and possibly EPC), we hypothesized that BPPs be able to increase the neovascularization repair of ischemic tissues by increasing the recruitment of EPCs and improving their functions.
Also, preconditioning of EPCs (from healthy individuals or patients with hypertension) ex vivo with prior to transplantation may be able to increase the potential vasculogênico / angiogenic these cells, thereby increasing the efficiency of the transplantation of EPCs . Thus, this research project has as main goal the development of innovative therapeutic strategies in cell therapy for revascularization of ischemic tissues by increasing the therapeutic efficiency of endothelial progenitor cells preconditioned ex vivo with peptides bradykinin potentiator. For our study will be used EPC derived from umbilical cord blood because they are more prevalent in blood volume as compared with equivalent EPC derived from peripheral blood from healthy adult individuals and to possess greater proliferative potential, among others. Furthermore, it is even possible to evaluate the ability of these peptides to rescue or enhance the properties vasculogenic / EPC angiogenic from patients with pre-eclampsia, one type of pregnancy-induced hypertension.
Researcher: Mayana Zatz / Institute: USP - SP
Neuromuscular disorders: from preclinical to clinical studies.
Since 2005 our group is investigating the potential of human adult stem-cells to differentiate in muscle cells both “in vitro” and “in vivo” with the aim to use them for cell therapy in neuromuscular disorders, in particular progressive muscular dystrophies or PMD ( Nunes et al., 2007, Secco et al., 2008 a,b;Secco et al., 2009; Vieira et al., 2007; Jazedje et al., 2009). However, before testing stem-cells as a therapy for PMD many questions needed to be addressed. What is the best way of delivery? What are the best cells to be injected? When to transplant? One or multiple injections? Is immunosuppression required? What directs homing? In order to address these questions we have injected human mesenchymal stromal cells (hMSCS) from dental pulp, adipose tissue and umbilical cord tissue in 3 animal models: SJL mice, LAMA2dy/2j mice and golden retriever muscular dystrophy (GRMD) dogs without immunosuppression. The transplant of hMSCs from dental pulp in GRMD dogs revealed that stem-cells delivery should be systemic ( Kerkis et al., 2008). hMSCs injections from both adipose tissue (AT) and umbilical cord tissue (UCT) into SJL mice and from UCT in LAMA2dy/2j mice showed that cells from both sources were able to reach the recipient muscle with a beneficial effect in injected animals (Vieira et al., 2008; 2010; Zucconi et al., 2011, Secco et al.,in press). Systemic injections of AT-hMSCs into GRMD dogs revealed the expression of human muscle proteins up to 6 months after the last injection with no apparent side effects 2 years after the last injection. (Vieira et al., 2011) These results suggest that stem-cell therapy might have a clinical application for different forms of muscular dystrophy regardless of specific mutations.
More recently, it was observed that the systemic injection of UC-MSCs together with IGF-1 had a beneficial effect in a severely affected murine model of congenital muscular dystrophy.
Therefore our aims now, which will be done as a collaborative study, are:
a) to repeat these experiments in other murine and dog models for muscular dystrophy in order to confirm the beneficial effect of combining MSCS and IGF-1 from umbilical cord and adipose tissue.
b) Repeat MSCs transplants in murine models for other conditions, mainly epilepsy and ALS
c) Start clinical trials with MSCs from adipose tissue in severely affected patients after approval by ethical committees.
Stem cells as a tool to enhance our comprehension on amyotrophic lateral sclerosis.
Amyotrophic Lateral Sclerosis (ALS),a devastating disorder, is the most common adult–onset motor neuron disease (MND) (Goodall and Morrison, 2006). Clinical findings include muscular atrophy and weakness, accompanied by fasciculation and spasticity, and a rapid and progressive degeneration of motor neurons in the cortex, brainstem and spinal cord. Approximately 90% of all ALS cases are sporadic (SALS) The remaining 10% familial forms (FALS) display mostly an autosomal dominant pattern of inheritance. More than ten different loci have been implicated in ALS.
Our group has mapped and identified the gene responsible for a dominant from of ALS, ALS8 which codes for the vesicle-associated membrane protein-associated protein-B/C (VAPB/C) or VAP-B (Nishimura et al., 2008a,b) . VAPs are typically ubiquitously expressed and localized to the endoplasmic reticulum (ER) and pre-Golgi intermediates They have been implicated in numerous cellular functions including the regulation of lipid transport and homeostasis, formation of presynaptic terminal, and unfolded protein responses . Hence, defects in many cellular processes are likely to be involved in the pathogenesis of ALS8. More recently we have derived IPS cells and motor neurons from ALS8 patients and normal relatives and showed that they have a decrease of VAP-B in fibroblasts, IPS cells and motor neurons (Mitne-Neto et al., 2011). These findings together with the observation that VAP-B is decreased in another mice model for ALS and in autopsies of sporadic ALS cases suggests a common loss of function mechanism for VAP-B in different forms of ALS .
More recently, three new genes for inherited forms of ALS have been identified: FUS (Yamashita et al. 2011), ubiquitilin (Deng et al., 2011) and a hexanucleotide repeat expansion in the C90RF72 gene (Renton et al., 2001;De Jesus-Hernandez M et al., 2011). This last gene could be responsible for 20% of sporadic cases.
Our group has ascertained a large sample of patients with ALS during recent years who were screened for the VAP-B and SOD1 genes. Therefore our plan now is:
a) Screen our patients for these additional 3 new genes
b) Collect fibroblasts and derive IPS and motor neurons from patients with known mutations
c) Analyze VAP-B in all samples
d) Verify if there is a common mechanism in patients carrying different mutations.
More recently, it was observed that the systemic injection of UC-MSCs together with IGF-1 had a beneficial effect in a severely affected murine model of congenital muscular dystrophy.
Therefore our aims now, which will be done as a collaborative study, are:
a) to repeat these experiments in other murine and dog models for muscular dystrophy in order to confirm the beneficial effect of combining MSCS and IGF-1 from umbilical cord and adipose tissue.
b) Repeat MSCs transplants in murine models for other conditions, mainly epilepsy and ALS
c) Start clinical trials with MSCs from adipose tissue in severely affected patients after approval by ethical committees.
Stem cells as a tool to enhance our comprehension on amyotrophic lateral sclerosis.
Amyotrophic Lateral Sclerosis (ALS),a devastating disorder, is the most common adult–onset motor neuron disease (MND) (Goodall and Morrison, 2006). Clinical findings include muscular atrophy and weakness, accompanied by fasciculation and spasticity, and a rapid and progressive degeneration of motor neurons in the cortex, brainstem and spinal cord. Approximately 90% of all ALS cases are sporadic (SALS) The remaining 10% familial forms (FALS) display mostly an autosomal dominant pattern of inheritance. More than ten different loci have been implicated in ALS.
Our group has mapped and identified the gene responsible for a dominant from of ALS, ALS8 which codes for the vesicle-associated membrane protein-associated protein-B/C (VAPB/C) or VAP-B (Nishimura et al., 2008a,b) . VAPs are typically ubiquitously expressed and localized to the endoplasmic reticulum (ER) and pre-Golgi intermediates They have been implicated in numerous cellular functions including the regulation of lipid transport and homeostasis, formation of presynaptic terminal, and unfolded protein responses . Hence, defects in many cellular processes are likely to be involved in the pathogenesis of ALS8. More recently we have derived IPS cells and motor neurons from ALS8 patients and normal relatives and showed that they have a decrease of VAP-B in fibroblasts, IPS cells and motor neurons (Mitne-Neto et al., 2011). These findings together with the observation that VAP-B is decreased in another mice model for ALS and in autopsies of sporadic ALS cases suggests a common loss of function mechanism for VAP-B in different forms of ALS .
More recently, three new genes for inherited forms of ALS have been identified: FUS (Yamashita et al. 2011), ubiquitilin (Deng et al., 2011) and a hexanucleotide repeat expansion in the C90RF72 gene (Renton et al., 2001;De Jesus-Hernandez M et al., 2011). This last gene could be responsible for 20% of sporadic cases.
Our group has ascertained a large sample of patients with ALS during recent years who were screened for the VAP-B and SOD1 genes. Therefore our plan now is:
a) Screen our patients for these additional 3 new genes
b) Collect fibroblasts and derive IPS and motor neurons from patients with known mutations
c) Analyze VAP-B in all samples
d) Verify if there is a common mechanism in patients carrying different mutations.
Researcher: Michel Eid Farah / Institute: UNIFESP-SP
Subretinal injection of stem cells from human bone marrow cells in transgenic mice with degeneration of photoreceptors.
The retinitis pigmentosa (RP) is a progressive degeneration of retinal photoreceptor cells (rods and cones) that starts at the periphery and moves toward the macula whose main symptoms are progressive difficulty closing night vision and visual field. So far there is no cure for RP, and there are treatments that are dubious or limited benefit in slowing disease progression, with needs for new forms of treatment for this pathology.
The objective of this study is to evaluate the survivability, integration, migration and differentiation of human stem cells from bone marrow transplanted into the subretinal space in an animal model genetically modified where there is degeneration of the RPE and photoreceptors in the retina of mice.
First will be collected human bone marrow in wing posterior iliac crest and preparation of human stem cells to obtain volume 1-2 ul of solution. To receive stem cell transplantation subretinal be used ten male C3H/HeJ mice, homozygous for the rd allele (rd / rd) at 2 months of life. In the right eye will be applying 1-2 ul solution containing hematopoietic progenitor cells in space subrretiniano 32 gauge needle in the left eye is injected with balanced salt solution. After 15 days of application, the animals are examined for functional evaluation eletroretinography and then sacrificed to prepare the material for immunohistochemistry with anti β-tubulin III, anti GFAP, anti-retinal pigment epithelium and anti-rhodopsin. Marking by TUNEL will be used to detect apoptotic cells.
Researcher: Nance Beyer Nardi / Institute: ULBRA - RS
Mapping of the niche regenerative in tissue damage and potential response of specific populations of adult stem cells.
The use of successful bone marrow and cord blood for treatment of hematologic malignancies, during the last 30 years led more recently to studies to use this same approach to other pathologies. Initial results suggested that stem cells (SC) present in the bone marrow had a great potential for repair of non-hematopoietic organs such as liver, brain and heart; the passage of these expectations for the scenario pre-clinical and clinical, however, proved that these observations could not be universally reproduced and showed the existence of methodological artifacts generating false results in early experiments. While expectations for use of the therapeutic potential of adult stem cells continue to encourage the scientific community, it becomes clear the need for more basic knowledge about these cells, for the design of experimental protocols consistent basis.
The multiple studies (basic, preclinical and clinical) that use adult stem cells are restricted, for the most part, to administration of cells such as mononuclear fraction of bone marrow or more purified subpopulations, such as mesenchymal stem cells, and evaluation of results in terms of correction of the lesion. These studies, which we call "type start-and-order", ignore the elements and events present in both the injury and administered in the cells, which are responsible for the outcome of the procedure.
One of the points that require further attention to better understanding of the biology of adult SC, is the question of the niche. Stem cells have their behavior determined by interactions with its microenvironment, or niche, which is composed of other cells, extracellular matrix and signaling factors. These interactions determine whether the cell remains quiescent or will enter into mitosis, giving rise in this case another SC and / or progenitors committed to differentiation. This concept is fundamental both for basic studies involving the maintenance of SC during in vitro cultivation, which requires the reproduction as faithful as possible to the physiological niche, and for its use in therapeutic procedures. If the target tissue is not a "proper injury", the cells will not recognize it as a niche in order to pursue their repair functions. Thus, equally or perhaps more important than knowing the cells to be used therapeutically, is to know the site of the lesion to be corrected.
The term "regenerative niche" has been used to describe the collection of cells present at an injured tissue. Despite producing factors that aim to attract regenerative cells, the lesion site also represents a microenvironment hostile, ischemic or necrotic, which may compromise the survival of transplanted cells. Thus, the understanding of the events working is key to successful intervention.
While other analyzes in the clinical setting are limited, in preclinical studies with the use of animal models is possible to evaluate the distribution of transplanted cells and the mechanisms responsible for the repair. Still, these points have been insufficiently explored. As a result, very little is known about the cellular and molecular events that occur at the injury site and populations of stem cells administered. This lack of knowledge hinders the design of more rational and effective therapeutic interventions, which remain highly empirical.
This project proposes to contribute to this definition, through the detailed mapping of cellular and molecular events involved in tissue injury in murine models, and the response potential of adult stem cells from murine and human to these elements. The selected models involve acute myocardial infarction (AMI) and peripheral ischemia, situations already well explored in studies of stem cell therapy such as "start-and-order" and for this reason, suitable for exploration in deeper level.
The project involves a large group of researchers to the appropriate division multiple tasks as the infrastructure of laboratories and experience of participants. The experimental approach basically involves the production of surgical lesions in C57BL/6 and the detailed and comprehensive analysis of elements present in the regeneration niche, when compared to healthy tissue. For this, serial sections are produced to analyze the morphology, cell phenotype, the degree of proliferation and apoptosis of cells in the sites of injury, and patterns of cytokines and growth factors produced. Analysis of these tissues by RT-PCR in real time inform on gene expression profiles. Identifying signs and profiles more interesting in terms of tissue regeneration, stem cell populations murine and human will be evaluated for their potential to respond to them.
Will be analyzed in the mononuclear fraction of bone marrow and human and murine populations of purified mesenchymal stem cells obtained from human bone marrow and bone marrow, liver, lungs and kidneys of mice C57BL/6 GFP-positive. The analyzes include gene expression profiles, production of ligands corresponding to the main signals from the injury site, basic features and karyotype of cultured MSCs, distribution pattern of murine cells in regenerative niche in situ or after systemic administration in a murine model of AMI, and treatment effect of MSCs in the repair of the lesion. It is expected that the combined analysis, in the same experimental system, of the niche and potential response of different cell populations, enable the identification of elements with greater therapeutic potential and mechanisms of action, which will allow the design of clinical protocols of cell therapy less empirical and with more experimental rationale.
Researcher: Niels Olsen Saraiva Câmara / Institute: USP-SP
Adult mesenchymal stem cells in regeneration and repair of acute and chronic renal injury: role of interaction with the immune system and inhibition of epithelial-mesenchymal transition.
In recent years, our research group has developed to characterize the inflammatory response observed in certain renal diseases in specific experimental models and in humans. Renal diseases have enormous social and economic impact with high morbidity and mortality for patients. Thus, the acquisition of new knowledge that will strengthen the knowledge about the pathogenesis of these diseases, and discovery of new strategies to improve renal dysfunction become necessary to improve patient survival.
Stem cells have great therapeutic potential to justify the extensive research in this area. In particular, the mesenchymal stem cells have characteristics that allow its use in clinical protocols, such as immunomodulation and lower allogenic effect. Using the model of ischemia-reperfusion injury (IRI), our laboratory demonstrated that treatment with mesenchymal stem cells from bone marrow and neural progenitors (neurospheres) was able to reverse the renal dysfunction. These cells reduced tissue inflammation by changing the pattern of immune response of Th1 (proinflammatory) into Th2 cells (anti-inflammatory). However, the mechanisms behind this modulation of the immune response remain unknown. Here, in this new project, we formulated a hypothesis that activation of mesenchymal stem cells by specific molecules induces the secretion of trophic factors.
The ability of these cells to polarize early immune response to a Th2 pattern, would be crucial in tissue regeneration. Furthermore, our preliminary data show that these cell group also reduce renal fibrosis in the remnant kidney model, suggesting that the modulation of immune response may be beneficial to sharply limit sclerosis tissue. Today, it is known that the tubulointerstitial fibrosis is a process result named epithelial-mesenchymal transition (EMT). Thus, we also believe that the mesenchymal stem cells can reverse or limit the damage by interfering with fibrotic EMT inducing dedifferentiation of fibroblasts.
In this project we wish to enhance the immunological mechanisms related to the regeneration of renal lesions in different models of cell damage in vitro by stem cells. Moreover, we extend our observations for models of chronic renal injury by demonstrating that these cells may modulate the EMT, totally innovative aspect in the literature. We will work with established models of acute and chronic renal injury and methodology of molecular and cellular biology to meet our objectives and hypotheses. We hope that all generated data will be published to facilitate a more rapid clinical translation, if shown the mechanism, safety and reproducibility of results.
Researcher: Oswaldo Tadeu Greco / Institute: IMC
Cell Therapy: Alternative treatment of clinical disorders refractory to conventional therapy.
Cardiovascular diseases are responsible for higher mortality at the time around the world and, by statistics, Brazil should have this strong growth rate in these next 10 years.
The increasing incidence of coronary disease risk factors, etiology inevitably this will be one that will contribute to greater increase in this index.
Even with advances in pharmacological and surgical procedures, vascular lesions do not respond to these behaviors, even with the growth of invasive ventricular dysfunction has increased significantly.
Therefore, I believe regenerative medicine, with implantation of autologous stem cells, has been presented as a good alternative therapy to repair the badly damaged myocardium.
The literature is already showing clinical and preclinical such conduct, both in cardiology and in clinical pulmonary and critical ischemia of lower limbs, has been a therapeutic option to consider in cases unresponsive to conventional practice.
Researcher: Oswaldo Keith Okamoto / Institute: UNIFESP-SP
Inhibition of tumorigenic property of human embryonic stem cells by gene silencing.
This scientific research project addresses an important aspect of biosecurity unresolved in the field of cell therapy and critical importance to the advancement of clinical applications with stem cells: the formation of tumors. This is a problem relevant to any type of stem cells. Nevertheless, given the striking association between the properties of pluripotency and tumorigenesis, this project will use as a model pre-established lines of human embryonic stem cells (hESC), the group available for use in basic research.
Recent studies from our group have identified genes with aberrant expression in neoplastic stem cells. Two of these genes, E2F2 and MELK, are involved in cell cycle control and embryonic development of mammals. Studies by independent groups indicate that E2F2 and MELK positively regulate expression of other genes important function in the process of self-renewal of stem cells, embryonic development and neoplastic transformation, including genes Bmi-1, c-Myc and b-Myb. Therefore, we will test whether the concomitant repression of genes E2F2 and MELK is able to reduce the proliferation of hESC and consequent formation of teratomas, based on its direct effects (on the cell cycle) and indirect (consequential inhibition of expression of various oncogenes target transcriptional network controlled by oncogenes and E2F2 regulated by signaling pathway MELK).
To this end, we will test the silencing of genes E2F2 and MELK system using a transient RNA of interference (iRNA), thus avoiding uncontrolled or permanent changes in the genome that can insert new mutations and to promote malignant transformation. A transient suppression of these genes would be appropriate to contain the first step involved in tumorigenesis hESC until the molecular programs involved in the proliferation of course be turned off in the presence of stimuli for cellular differentiation. In this context, an important exteriority of the project concerns the application of resources of nanobiotechnology aiming at greater efficiency and lower toxicity in the process of transfection of hESC. Search is ideally a tumorigenic inhibit condition that does not compromise the viability and pluripotency of hESC. This aspect will be assessed by proliferation assays, invasion and cell differentiation both in vitro and in vivo, the latter based on models of orthotopic and ectopic xeno-implants to assess teratoma formation and neural differentiation.
The scientific question of this project is far-reaching and immediate implications in the field of cell therapy. Studies along these lines would enable the application of different types of stem cells, including the newly characterized iPS cells (induced pluripotent stem cells) in diseases lacking effective treatment. The knowledge about the suppression of tumorigenesis of normal stem cells would also have implications in the fight against neoplastic stem cells, with a view to developing more effective treatments for cancer.
Researcher: Patricia Pranke / Institute: UFRGS
_The use of stem cells for tissue regeneration in the replacement of tissues in the treatment of laryngeal cancer and bone regeneration.
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Cell therapies for the use of mesenchymal stem cells (MSCs) have come to represent an alternative treatment in several areas of medicine. Laryngeal cancer, especially those in more advanced stages, determines a radical surgical procedure that interferes largely with the quality of life of individuals. The combination of techniques that produce biomaterials with stem cells also represents a promising strategy for bone regeneration.
The present study aims to use MSCs from two sources that are not invasive and are easily accessible: the pulp of deciduous teeth and umbilical cords, as well as their application for tissue regeneration. The MSCs, linked to the biomaterial, will be used to promote regeneration of bone tissue and cartilage in the larynx in animal models. The project aims to promote bone regeneration in rat models and the recovery of lost tissue after laryngectomy in pig models.
Cell therapies for the use of mesenchymal stem cells (MSCs) have come to represent an alternative treatment in several areas of medicine. Laryngeal cancer, especially those in more advanced stages, determines a radical surgical procedure that interferes largely with the quality of life of individuals. The combination of techniques that produce biomaterials with stem cells also represents a promising strategy for bone regeneration.
The present study aims to use MSCs from two sources that are not invasive and are easily accessible: the pulp of deciduous teeth and umbilical cords, as well as their application for tissue regeneration. The MSCs, linked to the biomaterial, will be used to promote regeneration of bone tissue and cartilage in the larynx in animal models. The project aims to promote bone regeneration in rat models and the recovery of lost tissue after laryngectomy in pig models.
Researcher: Patricia Rieken Macedo Rocco / Institute: UFRJ - RJ
Cell therapies for pulmonary and renal diseases prevalent: understanding the cellular and molecular mechanisms.
The use of cell therapies for cardiovascular diseases (Tura et al., 2007) and neurological (Mendez-Otero et al., 2007) presents promising horizons in Brazil and the same appears to outline the case of diabetes mellitus (Voltarelli et al., 2007). However, despite the high prevalence, severity and social cost of chronic lung and kidney diseases, as well as the few therapeutic alternatives (including allogeneic), the use of cell therapies in these diseases needs to be further investigated. This proposal is mainly aimed to investigate whether therapy with stem cells derived from bone marrow is a safe and viable option in the treatment of pulmonary and renal diseases of high prevalence, focusing on efforts to understand the cellular and molecular aspects of the interactions of these cells with the injured tissue and is proposing to establish templates for therapeutic application. Moreover, it will start Phase I of cell therapy in patients with silicosis (project already tested in animal models by our staff and approved by CONEP for human application). To this end, 22 researchers (12 laboratories, 6 Units of 3 Research Institutions) made a consortium in this CNPq announcement (CT-Saúde/MS/SCTIE/DECIT/MCT/CNPq Notice No. 17/2008), reissuing a combination of two years which resulted in the creation of an atmosphere and highly collaborative research in optimizing the use of common facilities and equipment.
Researcher: Paulo Roberto Slud Brofman / Institute: PUC - PR
In vitro and in vivo pro-angiogenic capacity of mesenchymal stem cells and progenitor cells treated with human endothelial nitric oxide donor.
Among the cardiovascular diseases, myocardial infarction, is the leading cause of death in the world population. Despite advances in medical and surgical constitutes also a serious public health problem. After infarction, the myocardium spontaneously try to regenerate damaged tissue by endogenous mobilization and migration of different cell types by specific pathways involving cytokines, changes in the extracellular matrix and adhesion molecules, but these actions are not sufficient for functional improvement. Furthermore, no clinically used drugs or procedure has proven effective in replacing the myocardial scar tissue-functional. Due to these factors there was a need for developing new therapeutic options to repair the injured myocardium.
In this context the cell therapy with the goal of using stem cells to treat cardiovascular diseases. Several preclinical studies and clinical trials have shown promising results of this therapeutic strategy in treating these serious diseases.
In particular endothelial progenitor cells and mesenchymal stem cells have been shown to be important regulators in the tissue repair process, the new vessel formation and regeneration of ischemic regions.
Studies have shown that treatment of mesenchymal stem cells with nitric oxide leads to increased expression of vascular endothelial growth factor, which is considered a central regulator of angiogenesis both in physiological and pathological conditions to exercise a dominant role in neovascularization and to differentiate into endothelial cells.
Our working hypothesis is that the treatment of mesenchymal stem cells with nitric oxide increase the pro-angiogenic paracrine ability and possibly the differentiation of these progenitor endothelial in endothelial cells. Thus, co-transplantation of mesenchymal stem cells treated with nitric oxide and endothelial progenitor cells have a beneficial effect greater than transplants of individual non-treated cultures.
Researcher: Radovan Borojevic / Institute: UFRJ - RJ
In vitro differentiation of cardiomyocytes from mesenchymal stem cells: induction of programmed gene expression.
Cellular therapies and bioengineered tissue grafting are new proposals for the treatment of ischemic heart disease and degenerative diseases. In most protocols under study, the cells used are derived from bone marrow, which can stimulate the regeneration by paracrine and / or participate in the formation of new myocardium by transdifferentiation. Both processes are random and difficult to control. The introduction of new cardiomyocytes, injected or implanted with biomaterials in bioengineering constructions, require a source of cardiomyocytes. Several studies work with cardiomyocytes derived from embryonic stem cells. Besides the problems of instability and tendency to form teratocarcinomas, these are allogeneic cells causing the anti-graft immune response.
Our proposal aims to develop the technology for mass production of human cardiomyocytes. The source cell will be the mesenchymal stem cells derived from bone marrow or subcutaneous adipose tissue of the patient. This option allows the use of autologous cells in the treatment of myocardial injury. The expression of cardiomyocyte phenotype is induced by the sequential introduction, combinatorial and set of genes by transfection of plasmids carrying genes selected. Its expression is limited in time, and do not integrate into the cell genome manipulated. At first, the introduction of genes into cells revert to embryonic tissue-specific differentiation of mesenchymal progenitors, generating induced pluripotent cells from adult cells. Then we will induce the expression of master genes that determine the commitment and full expression in the differentiation pathway in cardiomyocytes, following the order of activation of these genes in embryonic development. The complementary use of growth factors appropriate to each stage of differentiation will be studied. We will seek to optimize the process to make it feasible for clinical use, and verify the in vitro generation of structural and functional cardiomyocytes potentially used in treatment of heart disease.
Researcher: Rafael Linden / Institute: UFRJ - RJ
Mechanisms of cell cycle control of embryonic stem cells.
This project aims to examine the components and mechanisms of cell cycle control in the line of embryonic stem cells, undifferentiated or committed to neural progenitors, which may determine the phenotypic characteristics, in order to understand the behavior of these cells. In addition, we intend to analyze what happens in the cell cycle through inhibition of proliferation by expression of tumor suppressor genes p16INK4a and p21CIP and the process of induction of differentiation of embryonic stem cells into photoreceptors by gene expression NRL, crx and otx2, aiming at the future use of these cells in preclinical studies in murine model of retinitis pigmentosa (C3Hrd1 mice).
Researcher: Ricardo Ferreira Bento / Institute: USP-SP
Signaling and differentiation of cells of the organ of Corti: prospects for therapy of deafness.
Most hearing loss, congenital or acquired, results from the irreversible loss of cochlear hair cells or their neurons. The irreversibility of hearing loss in mammals is due to the inability to replace lost cells, either by returning to the absence of cell cycle or trans-differentiation of the epithelium of the inner ear. Clinically, the function of lost hair cells can be partially compensated by electrical stimulation of the auditory nerve via cochlear implants. However, alternative therapies with molecular and cellular bases need to be explored looking to improve hearing and quality of life of deaf patients.Because of the irreversibility of most cases of deafness, have tried to solve the molecular and cellular mechanisms related to hearing physiology and, consequently, its loss.
This research project includes the development of methodologies for research into the hearing with the following objectives: (i) to standardize procedures for obtaining and reintroduction of cells in the organ of Corti, in particular of the precursors of epithelial cells, hair, and (ii) standardize procedures and functional genetic modification of cultured cells of the organ of Corti. With these objectives in the long term, we intend to create experimental conditions fundamental to the development of future strategies of cell therapy and gene therapy for hearing loss.
Researcher: Ricardo Ribeiro dos Santos / Institute: FIOCRUZ - RJ
Cell therapy by autologous transplant of stem cells from bone marrow in patients with spinal cord injury.
Currently there is no effective treatment for the recovery of injured spinal cord. Based on experimental data from our group in dogs and cats with complete spinal cord injury, which indicated the safety and efficacy of transplantation of mesenchymal stem cells from autologous bone marrow in this type of injury, this project proposes the investigation of this therapy in patients with spinal cord injuries with more than 6 months of evolution, for which no one can offer at present no treatment capable of reversing this situation.
Based on experimental data from our group in dogs and cats with complete spinal cord injury, which indicated the safety and efficacy of transplantation of mesenchymal stem cells from autologous bone marrow in this type of injury, this project proposes the investigation of this therapy in patients with spinal cord injuries with more than 6 months of evolution, for which no one can offer at present no treatment capable of reversing this situation.
There will be a phase I clinical study to investigate whether administration of mesenchymal stem cells from autologous bone marrow is safe and causes the repair of spinal cord when injected into the lesion area in 20 patients with spinal cord injuries, complete injuries (Frankel A) chronic. The potential effectiveness of the method is evaluated by assessing the functional improvement (clinical, nuclear magnetic resonance and assessment of evoked potential).
Patients will undergo technical evaluation of the entire articular muscle structure, presence of contractures and hyperreflexia, sphincter control and bladder before the procedure. After surgery, the patient will undergo a daily physical therapy protocol. Hopefully, with this study, contribute to the development of new therapies based on cell transplantation for the treatment of chronic spinal cord injuries.
Researcher: Roberta Ferro de Godoy / Institute: UNB-DF
Stem cells in regeneration of the ulnar nerve in horses.
This research aims to verify the ulnar nerve regeneration after neurotomy and application of stem cells in equine females after surgery, comparing the conventional technique of suturing the epineurium with the application of silicone tubes with deposition of stem cells in regeneration of severed ulnar nerves. The procedure will be evaluated by quantifying the histological examination and histomorphometric pathological changes of the surgical site and peripheral nerve regeneration after conventional suture and deposition of stem cells from ulnar nerves sectioned female equine breed. Determined also by means of immunohistochemistry, axonal growth and formation of the myelin sheath by the expression of S-100 protein and neurofilament in ulnar nerve after section and suture or conventional deposition of stem cells in horses. And further assess through clinical specific exams of locomotor system, sensory and motor recovery of thoracic members and from the surgical site and the section of the ulnar nerves after treatment. Thus making it possible to verify the effectiveness of the use of stem cells in peripheral nerve regeneration in horses.
Researcher: Rosalia Mendez-Otero / Institute: UFRJ - RJ
Cell Therapy in Animal Models of Neurological Disease: basic and preclinical studies.
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Neurological diseases have high prevalence, mortality and social cost both in developed and developing countries. In Brazil, cerebrovascular diseases are the leading cause of death with approximately 90,000 deaths/year and effective pharmacological therapies are unavailable. In this context, stem cells have been considered as potential therapies and although promising, many questions must be answered before entering clinical practice.
In this project we will used several animal models of neurological diseases in order to investigate the possible therapeutic role of different types of stem cells. The results from the preclinical studies will be discussed with our collaborators in neurology to evaluate possible clinical studies.
Neurological diseases have high prevalence, mortality and social cost both in developed and developing countries. In Brazil, cerebrovascular diseases are the leading cause of death with approximately 90,000 deaths/year and effective pharmacological therapies are unavailable. In this context, stem cells have been considered as potential therapies and although promising, many questions must be answered before entering clinical practice.
In this project we will used several animal models of neurological diseases in order to investigate the possible therapeutic role of different types of stem cells. The results from the preclinical studies will be discussed with our collaborators in neurology to evaluate possible clinical studies.
Researcher: Rui Curi / Institute: USP-SP
Effect of therapy with muscle stem cells and mesenchymal stem cells in regeneration of skeletal muscle: possible modulation by fatty acids.
The possibility to manage a greater number of cells and to facilitate tissue regeneration represents a major challenge in the treatment of severe muscular lesions such as lacerations. This type of lesion shows incomplete regeneration and significant impairment of skeletal muscle function. However, this improvement in recovery from injuries due to cell therapy has been attributed to various mechanisms such as differentiation of stem cells into cells of damaged tissue, promoting angiogenesis, modulation of inflammatory phenomena and stimulation of spontaneous regeneration of the tissue through mechanisms paracrine. In this study we investigate the effect of omega 3, omega 6 and omega 9 in the mechanism of proliferation and differentiation of muscle stem cells murine and human in vitro and transplantation of muscle stem cells and mesenchymal stem cells from murine bone marrow in the recovery and regeneration of skeletal muscle function after experimental laceration. For this, we study the effect of cell therapy in the expression of molecular markers of myogenesis, angiogenesis and fibrogenesis in various periods of time after induction of muscle injury in syngeneic mice. The study will be complemented by histological analysis and testing of muscle function. In the second stage of labor, we investigate the possible modulatory role played by fatty acids in the effect of cell therapy, since there is evidence of the beneficial effect of some fatty acids in muscle differentiation in vitro.
Researcher: Samuel Goldenberg / Institute: ICC - PA
Epigenetic Analysis of mesenchymal stem cells from bone marrow in adipogenic induction.
The differentiation potential of adult stem cells is quite variable depending on the origin of the cells. Considering the role of epigenetic mechanisms in cell differentiation, we propose to investigate whether there is an epigenetic profile that features a greater or lesser potential for adipogenic differentiation of adult stem cells. Still, considering that the spatial organization of single genes has been related to their functional activity, another question raised is whether the spatial organization of genes characteristic of lineage-target (adipocyte) and stem cell-specific genes is modified with cell differentiation adult stem.
To answer these questions, the experimental model involves the adipogenic differentiation of mesenchymal stem cells isolated from bone marrow, a process already established in our group, and epigenetic analyzes, which consist of analyzes of histone modifications and DNA methylation . Thus we can establish a profile of epigenetic stem cell-specific genes and genes associated with lineage-target (adipocyte) in relation to modifications (acetylation and methylation) in histone and DNA methylation in the process of differentiation of cell cultures with high and low adipogenic potential. Obtaining this knowledge will contribute to the understanding of the mechanisms involved in the differentiation of adult stem cells.
Researcher: Sara Teresinha Olalla Saad / Institute: UNICAMP - SP
Action of mesenchymal cells derived from adipose tissue in the regeneration of knee cartilage lesions in rabbits.
The lesion of hyaline cartilage of the knee is a challenge to contemporary medicine. From the first descriptions up until this day, the inability of cartilage to repair itself normally through a spontaneous healing process is well known. Furthermore, the therapeutic measures adopted for the treatment of these lesions have so far been unsuccessful.
Tissue Engineering could be an answer to this problem. Numerous studies have demonstrated the possibility of obtaining cartilage, with similar characteristics to hyaline cartilage from mesenchymal cells. The development of methods for obtaining these cells from different sources, some with abundant material, such as adipose cells from obtained by liposuction, have stimulated these studies even further.
Another relevant aspect is the finding that the development of three-dimensional scaffolds of collagen and other substances have facilitated the formation of cartilage tissue.
In this project, using a model previously established at our institution, we obtained mesenchymal cells from adipose tissue collected during liposuction. After differentiation, collagen type II hydrogel (a biomaterial that mimics the microenvironment of the cartilage), obtained using a methodology developed at the Faculty of Medicine of Unicamp, Laboratory of Molecular Medicine, will be harvested with culture medium containing these cells. The complex type II collagen-mesenchymal cells are then implanted in the cartilage lesions produced in the cargo area of the knee of rabbits to observe the pattern of regeneration obtained. The tissue obtained with complex framework of collagen type II-mesenchymal cell (group IV) will be compared with the tissues formed in the spontaneous healing (group 1), collagen type II (group 2) and mesenchymal cells without scaffold (group 3). The aggregation and orientation of collagen fibrils in the hyaline cartilage of rabbit knees will be analyzed through Second Harmonic Generation (SHG). The effect of cryopreservation process with Dimethyl Sulfoxide (DMSO) upon the differentiation process of adipocyte tissue mesenchymal stem cells into chondrocyte lineages will also be evaluated by Second Harmonic Generation (SHG) microscopy.
Researcher: Sergio Paulo Bydlowski / Institute: USP - SP
Mesenchymal stem cells from human amniotic fluid - Characteristics of the processes of differentiation and use in animal models of spinal cord injury and chronic kidney disease.
Mesenchymal stem cells are considered multipotent cells that have the potential to differentiate into multiple cell lineages. They generated a great expectation as a potential source of cells for the development of therapeutic strategies, precisely because of its inherent capacity for self-renewal and differentiation. Is a rare population in the bone marrow of a human adult. Moreover, embryonic stem cells can give rise to any cell type more differentiated than the effective adult stem cells, which proliferate only for a limited number of generations, and whose response to signals differentiation decreases with each generation. The isolation of embryonic stem cells and their use are surrounded by debates and ethical issues. On the other hand, there is no serious issues ethical in obtaining adult hematopoietic stem cells, isolated with relative ease, but difficult to expand without occur differentiation.
An alternative to use of embryonic stem cells would be the use of stem cells from human amniotic fluid. In our laboratory we are more than two years studying the characteristics of these cells obtained around the 20th week of gestation, with good results, with funding obtained from the previous Notice of CNPq about stem cells.
This project aims to continue this study, deepening it, featuring the best cells, assessing the gene expression and protein both in undifferentiated cells as those that are differentiated. The behavior of cells that have undergone chondrogenic and osteogenic differentiation in scaffolds will be evaluated for further preclinical studies involving repair of bone and cartilage injuries. The conditions of various cryopreservation procedures in the maintenance of cellular features will be evaluated. Will also be surveyed for nesting characteristics of these stem cells in mice.
Will start preclinical studies with stem cells derived from amniotic fluid being applied in two experimental situations in rats. In a first, a model of chronic renal failure. In another, in a model of spinal cord injury, neurogenic induction of differentiation.
Researcher: Silvia Regina Batistuzzo de Medeiros / Institute: UFRN - RN
Human mesenchymal stem cells with karyotype aberrations show alterations in gene and chromosomal stability in the process of differentiation and senescence in vitro in the presence of titanium.
Human mesenchymal stem cells (hMSC) are good candidates for regenerative cell therapy, because of its ease of isolation and functional plasticity, but due to the low number of cells, it is necessary to undertake a considerable expansion in vitro prior to use. This can lead to aging and accumulation of oxidative damage and consequently in a decrease in the potential for proliferation and differentiation and lead to genetic instability. One application of hMSC is guided bone regeneration, and titanium is the most used material due to its biocompatibility and durability, but has been associated with the generation of oxidative stress. In previous studies, our laboratory has identified an inversion on chromosome 3 in a primary lines of existing hMSC. In the locus of inversion are located OGG1 genes, implicated in the repair of oxidative DNA damage and VHL that is a tumor suppressor. Thus, aim of this study is to investigate the potential susceptibility to malignant transformation of hMSC the umbilical vein with altered karyotype compared with hMSC with normal karyotype in different culture conditions in vitro in the presence and absence of titanium, whether or no plasma, and osteogenic factors before and after aging.
As a result of this project, we expect the formation of human resources, with multidisciplinary profile, in an recent and tip area in our country, strengthening graduate education in the Northeast, thereby reducing the technological imbalance between the Brazilian regions. As products is expected to generate knowledge about the relationship of the inversion in the short arm of chromosome 3 in the processes of: i) proliferation in vitro hMSC ii) osteogenesis and iii) senescence, always in the presence and / or absence of titanium modified by the plasma. It is also expected to understand the role of VHL and OGG1 protein in the cells of karyotype changes in the processes mentioned above.
Using a technique comprehensive differential proteomics, we hope to obtain a large protein expression pattern that can be used for understanding the operation of the cells in a quiescent state and proliferation, whether or not grown in the presence of titanium as well as cells with different karyotypes, and thus to contribute to understanding the biological processes of differentiation and stability of mesenchymal stem cells originating from umbilical vein. This may generate processes as the development of mechanisms of induction / inhibition of specific gene that provide differentiation and genetic stability of mesenchymal stem cells. Besides these indications, the results from this project will be of extreme scientific importance because it will contribute to improve the diagnosis of genetic stability of stem cells from umbilical cord before and after cell differentiation, the fundamental question for the transfer of technology for application in humans the replacement tissue.
Researcher: Stevens Rehen / Institute: ICB / UFRJ – RJ
Generation iPS cells from patients with mental disorders.
Schizophrenia has been defined as a neurodevelopmental disease that causes changes in the process of thoughts, perceptions and emotions, usually leading to a mental deterioration and affective blunting. Most of the knowledge about this mental disorder has been acquired from post-mortem brain analyses, from non-neural human cells or with animal models. Since induced pluripotent stem (iPS) cells are genetically identical to donor and capable of generating all cell types of an individual, including neurons, they have become an attractive model for the study of the human brain. Understanding the role of oxygen metabolism, both in the ill brain as well as throughout development, is of critical importance to elucidate pathophysiological mechanisms in psychiatric disorders such as schizophrenia. Our recent data provide evidence of metabolic changes, such as increased ROS levels and oxygen consumption, occurring during neurogenesis of iPS cells derived from schizophrenic patients when compared to controls. The use of iPS cells to study schizophrenia may provide a better understanding of the development of the disease, highlighting potential targets for treatment and drug screening.
Researcher: Tatiana Lobo Coelho de Sampaio / Institute: UFRJ - RJ
Cell therapy for chronic and acute spinal cord injuries.
The project proposed here aims to investigate the regenerative potential of three cell types, mesenchymal cells from adipose tissue, neural stem cells of the olfactory mucosa and olfactory ensheathing glial cells in acute and chronic spinal cord lesions induced experimentally in rats. The consortium of laboratories submitting the project consists of three groups who have worked together for four years in establishing the model for the study of spinal cord injuries (see patent application PI 0704128-4, INPI).
The project was built on the following assumptions: 1) preliminary data from the group showed that mesenchymal cells of adipose tissue and promote functional recovery after experimental spinal cord injury, 2) the olfactory mucosa and human adipose tissue are easily obtainable for autologous transplants, 3 ) both mesenchymal cells of adipose tissue and cells from the olfactory mucosa, have been tested in humans, with no reports of complications, 4) cell therapies proposed here can be easily combined with other therapies. This set of favorable factors coupled with previous experience of the group lead us to believe that the proposed project has great chances of success.
Researcher: Verônica Maria Morandi da Silva / Institute: UERJ - RJ
Role of cell adhesion in the recruitment of circulating endothelial progenitors to ischemic sites: in vitro and in vivo.
The modulation of endothelial cell adhesion to extracellular matrix molecules or fragments thereof (matricin), which interact with cellular receptors (integrin, proteoglycan, etc.). Affect cell survival and the angiogenic process. Our laboratory conducts studies on the role of thrombospondin-1 (TSP-1) in angiogenesis. In this project, the focus is the study of endothelial progenitor cells (EPCs), now considered essential for angiogenesis associated with the repair of ischemic lesions and cancer. We have demonstrated the effects of pro-angiogenic a 18 kDa fragment of TSP-1 (TSP18) and identify the two-sequence containing the consensus motif BBXB interaction with GAGs, that mimic the effect.
We also show that the pro-angiogenic effect of these peptides are mediated by sindecan-4, a heparan sulphate proteoglycan of the endothelial surface essential for the stabilization of focal contacts. The identification of sindecan-4 as a new target for modulating angiogenesis has led to interest in delving into different mechanisms of action of these molecules. We plan to establish the specificity of such regulation, characterized in endothelial cells already mature EPCs also isolated from peripheral blood of adults. Preliminary data indicate that pro-angiogenic peptides of TSP-1 can stimulate revascularization of ischemic sites. We intend to investigate whether the sindecan-4 is involved in this event and if it involves the recruitment of circulating endothelial progenitors. We will use experimental models based on endothelial cells transfected, to overexpress or silence the expression of sindecan-4 on their surfaces. The proliferation, differentiation, mobilization and incorporation of these cells to mature endothelium will be studied in vitro and in vivo (using models of experimental ischemia).
Researcher: Veronica Porto Carreiro de Vasconcellos Coelho / Institute: INCOR/USP - SP
Mesenchymal stem cells and regulatory T cells from human adipose tissue: interaction and immunoregulation.
Human mesenchymal cells (MSC) play an important role in hematopoiesis and, more recently, immunoregulatory capacity has been described. The adipose tissue derived from the MSC (Ad-MSC) may be an alternative source for the BM-MSC since it also can be easily expanded in vitro. Although these cells present a therapeutic potential, yet there are few studies evaluating the properties imunoreguladoras this cell type. The suppressive activity exerted by MSC appears to involve a variety of mechanisms that may depend both on the heterogeneity of MSC populations - yet little known - as the nature of the antigenic stimulus, the microenvironment and soluble factors present in it this way, the present study we have as the ability to compare.
We believe that a more global analysis of both molecules involved in immunoregulation, such as the induction of an inflammatory response, may provide us with important information about the many factors involved in the interaction of immune cells with the MSC. This will allow us to assess whether the balance between pro-inflammatory factors (IGNITES) and immunoregulatory (REGULATE) may contribute to the final outcome of the suppressive activity of MSC. Moreover, exploring the effect of interaction between the MSC derived from adipose tissue with CD4 + / CD25 + resident in the same microenvironment may contribute to a better understanding of mechanisms involved in communication between these two cell types and potential synergistic effects immunoregulatory.
Researcher: Walace Gomes Leal / Institute: UFPA - PA
Blocking microglial activation to maximize the neuroprotective effects of mesenchymal stem cells from bone marrow transplanted intravenously after experimental stroke.
One of the main hypotheses postulated for the beneficial effects of transplantation of stem cells from bone marrow is that this procedure would amplify the endogenous repair mechanisms. Moreover, it is known that microglial activation experimental damages during ischemia neurogenesis in regions of the CNS, such as the hippocampus. Moreover, recent studies suggest that the inflammatory environment, including microglial activation influences the therapeutic properties of stem cells transplanted. An important issue is the right time for the transplant after the occurrence of stroke. The same must be made in the acute phase, where the inflammatory process is more intense, or in the chronic phase, when the inflammatory response is more bland? It is possible that the therapeutic actions of transplanted stem cells are influenced by the inflammatory process. No studies that have investigated the influence of inflammation on the therapeutic actions of transplanted cells.
This project aims to investigate whether blockade of microglial cells in animals transplanted with stem cells from bone marrow induce neuroprotection, neurogenesis and motor recovery more effective, as assessed by histological techniques and behavioral analysis. Based on the above, this project will investigate the hypothesis that the treatment of ischemic animals with stem cells from bone marrow in conjunction with the blockade of microglial activation may induce tissue protection, neurogenesis and functional recovery more effective than simple cell therapy without blocking the inflammatory response. Our preliminary studies suggest that blocking microglial favors the therapeutic actions of bone marrow stem cells transplanted intravenously in ischemic motor cortex, creating a less inflammatory that promotes neuroprotection.
Researcher: William Dias Belangero / Institute: UNICAMP - SP
Study of the use of mesenchymal cells obtained from human adipose tissue scaffolds of the associated ß-tricalcium phosphate (ß-TCP), calcium phosphate, Ca3(PO4)2 and hydrogel of hyaluronic acid.
The mature mesenchymal cells correspond to a group of cells that can be obtained from the fatty tissue and have the ability in vitro to differentiate into bone tissue, which in turn may help treat bone defects caused by infection, trauma and tumors. Currently in clinical practice is used autologous different regions of the skeleton yet the donor source is insufficient for large losses and withdrawal can produce complications such as infection for the patient pain and paresthesias. The human adipose tissue (AT) obtained from liposuction can be processed to yield mesenchymal cells (MSC/AT) that under ideal growing conditions can differentiate into osteogenic cells with great potential use for the treatment of bone defects already mentioned. This new therapeutic approach generates the need to conduct preclinical studies to effectively evaluate the potential use of this source of MSC/AT.
Another important perspective that should be investigated is the use of scaffolds associated with MSC/AT to optimize the treatment of bone defects. Thus, this project aims to use osteogenic cells attached to scaffolds ß-Tricalcium phosphate (ß-TCP), calcium phosphate Ca3(PO4)2 and sodium hyaluronate hydrogel as carriers to resolve critical bone defects produced in the metaphyseal region proximal tibia of Wistar rats.
ß-tricalcium phosphate (ß-TCP), calcium phosphate Ca3(PO4)2 and hydrogels of hyaluronic acid were selected because they are biocompatible, biodegradable, porous with a chemical composition similar to that of human bone now used in clinical practice as inducer bone regeneration. The calcium phosphate Ca3(PO4)2 has similar characteristics to the ß-TCP, but are derived from papaya seeds (natural phosphates). Since the hydrogel of hyaluronic acid has different characteristics can be made with different morphologies and property specific to each type of application.
Researcher: Wilson Savino / Institute: FIOCRUZ - RJ
Cell therapy in muscular dystrophies.
The transplantation of skeletal muscle precursor cells has been seen as a potential strategy to treat muscular dystrophies.
However, despite having been used in different designs, is still low transfer efficiency of myogenic precursor and maintenance of the same receptor in muscle tissue, following the procedures for cell therapy. Accordingly, on the one hand we have the obstacle course the adaptive immune response of the individual receiving the transplant, and on the other hand the actual muscle tissue microenvironment where the cells are transplanted. In this project, we intend to address these two aspects, using model of cell therapy with human myogenic precursors injected into immunodeficient mice or immunocompetent.
With respect to the molecular mechanisms intrinsic to the muscle tissue and involved in survival and dispersal of human myogenic precursors after inoculation in immunodeficient animals, we want to determine:
1) The term intramuscular injection of extracellular matrix molecules, chemokines, cytokines and extracellular matrix metalloproteases that can be involved in the inflammatory cell influx during the handle of transplanted cells for cell therapy;
2) The expression pattern of pro-apoptotic molecules (including the process of cell death) expressed by human myoblasts before and after the procedure for cell therapy;
3) The effect of molecules of ECM molecules, associated or not with chemokines and growth factors on ex vivo migration of myogenic precursors;
4) The effect of matrix metalloproteinases on the ex vivo migration of myogenic precursors induced by ECM molecules, chemokines associated or not;
5) The degree of dispersion and myogenic precursors of human apoptosis in vivo, within the muscle receptor mice after treatment with ECM molecules, in combination or not with chemokines, particularly CXCL12.
A second therapeutic approach to improve the conditions of transplantation of human myogenic precursors in mice is induced in immunocompetent recipient animals, immune tolerance to the cells that are transplanted into the muscle tissue. Therefore, we intend to test two different strategies to generate central tolerance.
a) intrathymic injection of human myoblasts (or extracts from these cells) in normal mice immediately subjected to lethal irradiation and transferred totipotent precursors in bone marrow from syngeneic animals. In this situation, the animals are to reconstitute his repertoire of T cells in the thymus, in the presence of molecules derived from human, deletion of inducing T cell clones expressing the antigen receptor specific for epitopes of these molecules. Thus, by means of negative selection, we can expect a significant decrease of immune response against cells that are later transplanted into the muscle receptor.
b) lethal irradiation of animals, followed by injection of bone marrow precursors with human myoblasts. Once arriving myogenic precursors in the thymus, this second protocol also expected to generate central tolerance, and therefore immune conditions permissive for subsequent xenogenic transplantation of human myogenic precursors.
That protocol gives better results in terms of tolerance will be used subsequently in mdx mice, followed by an analysis of muscle tissue after transplantation of human cells.
We have no doubt that the accumulated knowledge about the factors influencing on survival, growth, dispersion and differentiation of these precursors in vivo is of great importance for future design of new therapeutic trials. Furthermore, the data on the generation of immune tolerance will allow a more concrete theoretical basis for delineate therapeutic protocols to be tested in immunocompetent individuals. Taken together, these two conceptual approaches represent an innovation in terms of cell therapy protocols aiming at the future correction of muscular dystrophy.
In conclusion, the possibility of approaching the one hand issues intrinsic biological the ecological niche receiving transplantation of human myogenic precursors and on the other hand the induction of immunological tolerance to such cells should lead to significant contribution to obtain greater efficiency of transplantation of myogenic precursors in immunocompetent individuals.
Researcher: Rubens Camargo Siqueira / Institute: USP - Ribeirão Preto
Use of stem cells derived from bone marrow for the treatment of retinal disease.
There are several types retinopathies that may result in blindness. Some have a genetic origin and affect individuals from birth, others are chronic degenerative diseases such as diabetic retinopathy, with a higher incidence in the population over 50 years.
Hereditary retinal dystrophies such as retinitis pigmentosa affects 1 in 3000 individuals and are characterized by progressive night blindness, visual field loss, optic atrophy, arteriolar attenuation, altered vascular permeability and progressive loss of central vision to complete blindness. Molecular genetic analysis of these diseases has identified over 110 different genes have been identified thus far responsible for triggering these entities that have no treatment until the moment.
Another important disease is age-related macular degeneration (AMD) is the leading cause of blindness in people over 55 years. Its prevalence increases with age affecting about 8.5 to 27.9% of the population over 75 years. It has been observed that the incidence of this disease has increased in recent decades in the order of 30 to 40%, in spite of eye diseases such as cataracts and glaucoma, which reach the same population group, have shown apparently reduced their records.
Although the etiology has not yet been determined, it has been shown that environmental and genetic factors may be involved. An oxidative insult that results in death of photoreceptors in the macula could be the genesis of the process.
Finally the retinal vascular diseases that lead to ischaemia, a condition which does not have any treatment to reverse the functional retinal tissue leading to blindness. Among the causes of ischemic retinopathy, diabetic retinopathy is the most common worldwide.
According to the World Health Organization more than 220 million people have diabetes.
The absence of effective treatments for most retinal degenerative diseases associated with increased longevity and incidence of diabetes, this number will only grow.
We are conducting three clinical trials authorized by the National Research Ethics (CONEP) using stem cells derived from bone marrow to treat the following diseases:
1) Hereditary Degenerations of the retina (retinitis pigmentosa) - Started in 2009
2) age-related macular degeneration (AMD) - Started in 2011
3) ischemic retinopathy (diabetic retinopathy) - Started in 2011.
Researcher: Arnaldo Rodrigues dos Santos Jr / Institute: UFABC
Use of Biomaterials and mesenchymal stem cells in tissue regeneration and engineering applied to traumatology.
The Tissue Engineering refers to the combination of methods Cell and Tissue Biology, Engineering and Surgery, to elucidate structure-function relationships in normal and damaged tissues aiming at repairing or replacing them. These methodology may be used in conjunction with the use of biomaterials in form of scaffolds developed to fill and repair damaged tissues.
Among the materials which can be used to produce scaffolds are bioresorbable polymers, with when implanted, are eliminated from the body by physiological pathways. Stem cells are seen as a possible solution for cell therapy and tissue regeneration. The mesenchymal stem cells have a great capacity for proliferation and differentiation, and are obtained and used in a satisfactory way.
We aiming the use of mesenchymal stem cells in induction of differentiation in to a specific cell type, stimulating their proliferation and inserting these cells in damaged tissue. To use mesenchymal stem cells, makes it necessary to standardize their methods of production, maintenance and specific differentiation, associated with the need for preclinical and clinical trials to effectively evaluate their therapeutic potential. From these studies, we propose to evaluate scaffolds designed for use in patients with severe problems tissue injury. Our investigation is to standardize the methodologies for obtaining and cell culture, their induction to differentiation of mesenchymal stem cells, the use of biocompatibility tests for of biomaterials focused on the construction of scaffolds for tissue repair, basically focusing bone and skin repair.
Among the materials which can be used to produce scaffolds are bioresorbable polymers, with when implanted, are eliminated from the body by physiological pathways. Stem cells are seen as a possible solution for cell therapy and tissue regeneration. The mesenchymal stem cells have a great capacity for proliferation and differentiation, and are obtained and used in a satisfactory way.
We aiming the use of mesenchymal stem cells in induction of differentiation in to a specific cell type, stimulating their proliferation and inserting these cells in damaged tissue. To use mesenchymal stem cells, makes it necessary to standardize their methods of production, maintenance and specific differentiation, associated with the need for preclinical and clinical trials to effectively evaluate their therapeutic potential. From these studies, we propose to evaluate scaffolds designed for use in patients with severe problems tissue injury. Our investigation is to standardize the methodologies for obtaining and cell culture, their induction to differentiation of mesenchymal stem cells, the use of biocompatibility tests for of biomaterials focused on the construction of scaffolds for tissue repair, basically focusing bone and skin repair.
