Exploring the Role of Soluble Factors Associated with Immune Regulatory Properties of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are characterized as multipotent stromal cells with the capacity for both self-renewal and differentiation into mesodermal cell lineages. MSCs also have a fibroblast-like phenotype and can be isolated from several tissues. In recent years, researchers have found that MSCs secrete several soluble factors that exert immunosuppressive effects by modulating both innate (macrophages, dendritic and NK cells) and adaptive (B cells and CD4+ and CD8+ T cells) immune responses. This review summarizes the principal trophic factors that are related to immune regulation and secreted by MSCs under both autoimmune and inflammatory conditions. The understanding of mechanisms that regulate immunity in MSCs field is important for their future use as a novel cellular-based immunotherapy with clinical applications in several diseases.

Morphological characterization of the progenitor blood cells in canine and feline umbilical cord

The umbilical cord blood (UCB) is an important source of hematopoietic stem cells with great deal of interest in regenerative medicine. The UCB cells have been extensively studied as an alternative to the bone marrow transplants. The challenge is to define specific methods to purify and characterize these cells in different animal species. This study is aimed at morphological characterization of progenitor cells derived from UCB highlighting relevant differences with peripheral blood of adult in dog and cats. Therefore, blood was collected from 18 dogs and 5 cats' umbilical cords from fetus in various developmental stages. The mononuclear cells were separated using the gradient of density Histopaque-1077. Characterization of CD34+ cells was performed by flow cytometric analysis and transmission electron microscopy. Granulocytes (ancestry of the basophiles, eosinophiles, and neutrophiles) and agranulocytes (represented by immature lymphocytes) were identified. We showed for the first time the ultrastructural features of cat UCB cells. Microsc. Res. Tech. 75:766770, 2012. (C) 2011 Wiley Periodicals, Inc.

Mesenchymal stem cells with rhBMP-2 inhibits the growth of canine osteosarcoma cells

Background: The bone morphogenetic proteins (BMPs) belong to a unique group of proteins that includes the growth factor TGF-beta. BMPs play important roles in cell differentiation, cell proliferation, and inhibition of cell growth. They also participate in the maturation of several cell types, depending on the microenvironment and interactions with other regulatory factors. Depending on their concentration gradient, the BMPs can attract various types of cells and act as chemotactic, mitogenic, or differentiation agents. BMPs can interfere with cell proliferation and the formation of cartilage and bone. In addition, BMPs can induce the differentiation of mesenchymal progenitor cells into various cell types, including chondroblasts and osteoblasts. The aim of this study was to analyze the effects of treatment with rhBMP-2 on the proliferation of canine mesenchymal stem cells (cMSCs) and the tumor suppression properties of rhBMP-2 in canine osteocarcoma (OST) cells. Osteosarcoma cell lines were isolated from biopsies and excisions of animals with osteosarcoma and were characterized by the Laboratory of Biochemistry and Biophysics, Butantan Institute. The mesenchymal stem cells were derived from the bone marrow of canine fetuses (cMSCs) and belong to the University of Sao Paulo, College of Veterinary Medicine (FMVZ-USP) stem cell bank. After expansion, the cells were cultured in a 12-well Transwell system; cells were treated with bone marrow mesenchymal stem cells associated with rhBMP2. Expression of the intracytoplasmic and nuclear markers such as Caspase-3, Bax, Bad, Bcl-2, Ki-67, p53, Oct3/4, Nanog, Stro-1 were performed by flow citometry. Results: We evaluated the regenerative potential of in vitro treatment with rhBMP-2 and found that both osteogenic induction and tumor regression occur in stem cells from canine bone marrow. rhBMP-2 inhibits the proliferation capacity of OST cells by mechanisms of apoptosis and tumor suppression mediated by p53. Conclusion: We propose that rhBMP-2 has great therapeutic potential in bone marrow cells by serving as a tumor suppressor to increase p53 and the pro-apoptotic proteins Bad and Bax, as well as by increasing the activity of phosphorylated caspase 3. Study design: Canine bone marrow mesenchymal stem cells associated with rhBMP2 in canine osteosarcoma treatment: "in vitro" study

Kinin-B2 Receptor Activity Determines the Differentiation Fate of Neural Stem Cells

Bradykinin is not only important for inflammation and blood pressure regulation, but also involved in neuromodulation and neuroprotection. Here we describe novel functions for bradykinin and the kinin-B2 receptor (B2BkR) in differentiation of neural stem cells. In the presence of the B2BkR antagonist HOE-140 during rat neurosphere differentiation, neuron-specific beta 3-tubulin and enolase expression was reduced together with an increase in glial protein expression, indicating that bradykinin- induced receptor activity contributes to neurogenesis. In agreement, HOE-140 affected in the same way expression levels of neural markers during neural differentiation of murine P19 and human iPS cells. Kinin-B1 receptor agonists and antagonists did not affect expression levels of neural markers, suggesting that bradykinin-mediated effects are exclusively mediated via B2BkR. Neurogenesis was augmented by bradykinin in the middle and late stages of the differentiation process. Chronic treatment with HOE-140 diminished eNOS and nNOS as well as M1-M4 muscarinic receptor expression and also affected purinergic receptor expression and activity. Neurogenesis, gliogenesis, and neural migration were altered during differentiation of neurospheres isolated from B2BkR knock-out mice. Whole mount in situ hybridization revealed the presence of B2BkR mRNA throughout the nervous system in mouse embryos, and less beta 3-tubulin and more glial proteins were expressed in developing and adult B2BkR knock-out mice brains. As a underlying transcriptional mechanism for neural fate determination, HOE-140 induced up-regulation of Notch1 and Stat3 gene expression. Because pharmacological treatments did not affect cell viability and proliferation, we conclude that bradykinin-induced signaling provides a switch for neural fate determination and specification of neurotransmitter receptor expression.

Altered Oxygen Metabolism Associated to Neurogenesis of Induced Pluripotent Stem Cells Derived From a Schizophrenic Patient

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. Studies have shown altered cell respiration and oxidative stress response in schizophrenia; however, most of the knowledge has been acquired from postmortem brain analyses or from nonneural cells. Here we describe that neural cells, derived from induced pluripotent stem cells generated from skin fibroblasts of a schizophrenic patient, presented a twofold increase in extramitochondrial oxygen consumption as well as elevated levels of reactive oxygen species (ROS), when compared to controls. This difference in ROS levels was reverted by the mood stabilizer valproic acid. Our model shows evidence that metabolic changes occurring during neurogenesis are associated with schizophrenia, contributing to a better understanding of the development of the disease and highlighting potential targets for treatment and drug screening.

Expression of Pancreatic Endocrine Markers by Mesenchymal Stem Cells From Human Adipose Tissue

Mesenchymal stem cells (MSCs) from human adipose tissue have a great potential for use in cell therapy due to their ease of isolation, expansion, and differentiation, besides the relative acceptance from the ethical point of view. Our intention was to isolate and promote in vitro expansion and differentiation of MSCs from human adipose tissue into cells with a pancreatic endocrine phenotype. Human adipose tissue obtained from patients undergoing abdominal dermolipectomy was digested with type I collagenase. MSCs isolated by plastic adherence and characterized by cytochemistry and FACS were expanded in vitro. MSC differentiation into an endocrine phenotype was induced over 2 to 4 months with high glucose (25 mmol/L) media containing nicotinamide, exendin-4, and 2-mercaptoethanol. Insulin and glucagon expressions were analyzed by immunofluorescence. Cells isolated from human adipose tissue and expanded in vitro expressed MSC markers as confirmed by FACS and cytochemistry. Insulin but not glucagon production by differentiated cells was demonstrated by irnmunofluorescence. MSCs isolated from human adipose tissue were induced to differentiate in vitro into an endocrine phenotype that expressed insulin

Risks, Benefits, and Therapeutic Potential of Hematopoietic Stem Cell Transplantation for Autoimmune Diabetes

Type 1 diabetes mellitus is a chronic disease that results from the autoimmune response against pancreatic insulin producing beta cells. Apart of several insulin regimens, since the decade of 80s various immunomodulatory regimens were tested aiming at blocking some steps of the autoimmune process against beta cell mass and at promoting beta cell preservation. In the last years, some independent research groups tried to cure type 1 diabetes with an "immunologic reset" provided by autologous hematopoietic stem cell transplantation in newly diagnosed patients, and the majority of patients became free form insulin with increasing levels of C-peptide along the time. In this review, we discuss the biology of hematopoietic stem cells and the possible advantages and disadvantages related to the high dose immunosuppression followed by autologous hematopoietic stem cell transplantation.

A quantitative proteomic and transcriptomic comparison of human mesenchymal stem cells from bone marrow and umbilical cord vein

Human mesenchymal stem cells (hMSCs) are adult multipotent cells that have high therapeutic potential due to their immunological properties. They can be isolated from several different tissues with bone marrow (BM) being the most common source. Because the isolation procedure is invasive, other tissues such as human umbilical cord vein (UCV) have been considered. However, their interchangeability remains unclear. In the present study, total protein extracts of BM-hMSCs and UCV-hMSCs were quantitatively compared using gel-LC-MS/MS. Previous SAGE analysis of the same cells was re-annotated to enable comparison and combination of these two data sets. We observed a more than 63% correlation between proteomic and transcriptomic data. In silico analysis of highly expressed genes in cells of both origins suggests that they can be modulated by microRNA, which can change protein abundance. Our results showed that MSCs from both tissues shared high similarity in metabolic and functional processes relevant to their therapeutic potential, especially in the immune system process, response to stimuli, and processes related to the delivery of the hMSCs to a given tissue, such as migration and adhesion. Hence, our results support the idea that the more accessible UCV could be a potentially less invasive source of MSCs.

In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration

Support structures for dermal regeneration are composed of biodegradable and bioresorbable polymers, animal skin or tendons, or are bacteria products. The use of such materials is controversial due to their low efficiency. An important area within tissue engineering is the application of multipotent mesenchymal stromal cells (MSCs) to reparative surgery. The combined use of biodegradable membranes with stem cell therapy may lead to promising results for patients undergoing unsuccessful conventional treatments. Thus, the aim of this study was to test the efficacy of using membranes composed of anionic collagen with or without the addition of hyaluronic acid (HA) as a substrate for adhesion and in vitro differentiation of bone marrow-derived canine MSCs. The benefit of basic fibroblast growth factor (bFGF) on the differentiation of cells in culture was also tested. MSCs were collected from dog bone marrow, isolated and grown on collagen scaffolds with or without HA. Cell viability, proliferation rate, and cellular toxicity were analyzed after 7 days. The cultured cells showed uniform growth and morphological characteristics of undifferentiated MSCs, which demonstrated that MSCs successfully adapted to the culture conditions established by collagen scaffolds with or without HA. This demonstrates that such scaffolds are promising for applications to tissue regeneration. bFGF significantly increased the proliferative rate of MSCs by 63% when compared to groups without the addition of the growth factor. However, the addition of bFGF becomes limiting, since it has an inhibitory effect at high concentrations in culture medium.

Vascular wall stem cells. Selection and conditioning of progenitors useful for cell therapy. A pathological case study

The arterial wall contains MSCs with mesengenic and angiogenic abilities. These multipotent precursors have been isolated from variously-sized human adult segments, belying the notion that vessel wall is a relatively quiescent tissue. Recently, our group identified in normal human arteries a vasculogenic niche and subsequently isolated and characterized resident MSCs (VW-MSCs) with angiogenic ability and multilineage potential. To prove that VW-MSCs are involved in normal and pathological vascular remodeling, we used a long-term organ culture system; this method was of critical importance to follow spontaneous 3-D vascular remodeling without any influence of blood cells. Next we tried to identify and localize in situ the VW-MSCs and to understand their role in the vascular remodeling in failed arterial homografts. Subsequently, we isolated this cell population and tested in vitro their multilineage differentiation potential through immunohistochemical, immunofluorescence, RT-PCR and ultrastructural analysis. From 25-30cm2 of each vascular wall homograft sample, we isolated a cell population with MSCs properties; these cells expressed MSC lineage molecules (CD90, CD44, CD105, CD29, CD73), stemness (Notch-1, Oct-4, Sca-1, Stro-1) and pericyte markers (NG2) whilst were negative for hematopoietic and endothelial markers (CD34, CD133, CD45, KDR, CD146, CD31 and vWF). MSCs derived from failed homografts (H-MSCs) exhibited adipogenic, osteogenic and chondrogenic potential but scarce propensity to angiogenic and leiomyogenic differentiation. The present study demonstrates that failed homografts contain MSCs with morphological, phenotypic and functional MSCs properties; H-MSCs are long-lived in culture, highly proliferating and endowed with prompt ability to differentiate into adipocytes, osteocytes and chondrocytes; compared with VW-MSCs from normal arteries, H-MSCs show a failure in angiogenic and leiomyogenic differentiation. A switch in MSCs plasticity could be the basis of pathological remodeling and contribute to aneurysmal failure of arterial homografts. The study of VW-MSCs in a pathological setting indicate that additional mechanisms are involved in vascular diseases; their knowledge will be useful for opening new therapeutic options in cardiovascular diseases.

3D nanostructured microcarriers for cell therapy in regenerative medicine

Supercritical Emulsion Extraction technology (SEE-C) was proposed for the production of poly-lactic-co-glycolic acid microcarriers. SEE-C operating parameters as pressure, temperature and flow rate ratios were analyzed and the process performance was optimized in terms of size distribution and encapsulation efficiency. Microdevices loaded with bovine serum insulin were produced with different sizes (2 and 3 µm) or insulin charges (3 and 6 mg/g) and with an encapsulation efficiency of 60%. The microcarriers were characterized in terms of insulin release profile in two different media (PBS and DMEM) and the diffusion and degradation constants were also estimated by using a mathematical model. PLGA microdevices were also used in a cultivation of embryonic ventricular myoblasts (cell line H9c2 obtained from rat) in a FBS serum free medium to monitor cell viability and growth in dependence of insulin released. Good cell viability and growth were observed on 3 µm microdevices loaded with 3 mg/g of insulin. PLGA microspheres loaded with growth factors (GFs) were charged into alginate scaffold with human Mesenchimal Steam Cells (hMSC) for bone tissue engineering with the aim of monitoring the effect of the local release of these signals on cells differentiation. These “living” 3D scaffolds were incubated in a direct perfusion tubular bioreactor to enhance nutrient transport and exposing the cells to a given shear stress. Different GFs such as, h-VEGF, h-BMP2 and a mix of two (ratio 1:1) were loaded and alginate beads were recovered from dynamic (tubular perfusion system bioreactor) and static culture at different time points (1st, 7th, 21st days) for the analytical assays such as, live/dead; alkaline phosphatase; osteocalcin; osteopontin and Van Kossa Immunoassay. The immunoassay confirmed always a better cells differentiation in the bioreactor with respect to the static culture and revealed a great influence of the BMP-2 released in the scaffold on cell differentiation.

In vitro characterisation and expansion of human regulatory T cells for their in vivo application in the induction of tolerance in haematopoietic stem cell and solid organ transplantation

Solid organ transplantation (SOT) is considered the treatment of choice for many end-stage organ diseases. Thus far, short term results are excellent, with patient survival rates greater than 90% one year post-surgery, but there are several problems with the long term acceptance and use of immunosuppressive drugs. Hematopoietic Stem Cells Transplantation (HSCT) concerns the infusion of haematopoietic stem cells to re-establish acquired and congenital disorders of the hematopoietic system. The main side effect is the Graft versus Host Disease (GvHD) where donor T cells can cause pathology involving the damage of host tissues. Patients undergoing acute or chronic GvHD receive immunosuppressive regimen that is responsible for several side effects. The use of immunosuppressive drugs in the setting of SOT and GvHD has markedly reduced the incidence of acute rejection and the tissue damage in GvHD however, the numerous adverse side effects observed boost the development of alternative strategies to improve the long-term outcome. To this effect, the use of CD4+CD25+FOXP3+ regulatory T cells (Treg) as a cellular therapy is an attractive approach for autoimmunity disease, GvHD and limiting immune responses to allograft after transplantation. Treg have a pivotal role in maintaining peripheral immunological tolerance, by preventing autoimmunity and chronic inflammation. Results of my thesis provide the characterization and cell processing of Tregs from healthy controls and patients in waiting list for liver transplantation, followed by the development of an efficient expansion-protocol and the investigation of the impact of the main immunosuppressive drugs on viability, proliferative capacity and function of expanded cells after expansion. The conclusion is that ex vivo expansion is necessary to infuse a high Treg dose and although many other factors in vivo can contribute to the success of Treg therapy, the infusion of Tregs during the administration of the highest dose of immunosuppressants should be carefully considered.

Design methods and geometric modelling of scaffolds for bone tissue engineering

Every year, thousand of surgical treatments are performed in order to fix up or completely substitute, where possible, organs or tissues affected by degenerative diseases. Patients with these kind of illnesses stay long times waiting for a donor that could replace, in a short time, the damaged organ or the tissue. The lack of biological alternates, related to conventional surgical treatments as autografts, allografts, e xenografts, led the researchers belonging to different areas to collaborate to find out innovative solutions. This research brought to a new discipline able to merge molecular biology, biomaterial, engineering, biomechanics and, recently, design and architecture knowledges. This discipline is named Tissue Engineering (TE) and it represents a step forward towards the substitutive or regenerative medicine. One of the major challenge of the TE is to design and develop, using a biomimetic approach, an artificial 3D anatomy scaffold, suitable for cells adhesion that are able to proliferate and differentiate themselves as consequence of the biological and biophysical stimulus offered by the specific tissue to be replaced. Nowadays, powerful instruments allow to perform analysis day by day more accurateand defined on patients that need more precise diagnosis and treatments.Starting from patient specific information provided by TC (Computed Tomography) microCT and MRI(Magnetic Resonance Imaging), an image-based approach can be performed in order to reconstruct the site to be replaced. With the aid of the recent Additive Manufacturing techniques that allow to print tridimensional objects with sub millimetric precision, it is now possible to practice an almost complete control of the parametrical characteristics of the scaffold: this is the way to achieve a correct cellular regeneration. In this work, we focalize the attention on a branch of TE known as Bone TE, whose the bone is main subject. Bone TE combines osteoconductive and morphological aspects of the scaffold, whose main properties are pore diameter, structure porosity and interconnectivity. The realization of the ideal values of these parameters represents the main goal of this work: here we'll a create simple and interactive biomimetic design process based on 3D CAD modeling and generative algorithmsthat provide a way to control the main properties and to create a structure morphologically similar to the cancellous bone. Two different typologies of scaffold will be compared: the first is based on Triply Periodic MinimalSurface (T.P.M.S.) whose basic crystalline geometries are nowadays used for Bone TE scaffolding; the second is based on using Voronoi's diagrams and they are more often used in the design of decorations and jewellery for their capacity to decompose and tasselate a volumetric space using an heterogeneous spatial distribution (often frequent in nature). In this work, we will show how to manipulate the main properties (pore diameter, structure porosity and interconnectivity) of the design TE oriented scaffolding using the implementation of generative algorithms: "bringing back the nature to the nature".

Toward cell therapy using placenta-derived cells: disease mechanisms, cell biology, preclinical studies, and regulatory aspects at the round table

Among the many cell types that may prove useful to regenerative medicine, mounting evidence suggests that human term placenta-derived cells will join the list of significant contributors. In making new cell therapy-based strategies a clinical reality, it is fundamental that no a priori claims are made regarding which cell source is preferable for a particular therapeutic application. Rather, ongoing comparisons of the potentiality and characteristics of cells from different sources should be made to promote constant improvement in cell therapies, and such comparisons will likely show that individually tailored cells can address disease-specific clinical needs. The principle underlying such an approach is resistance to the notion that comprehensive characterization of any cell type has been achieved, neither in terms of phenotype nor risks-to-benefits ratio. Tailoring cell therapy approaches to specific conditions also requires an understanding of basic disease mechanisms and close collaboration between translational researchers and clinicians, to identify current needs and shortcomings in existing treatments. To this end, the international workshop entitled "Placenta-derived stem cells for treatment of inflammatory diseases: moving toward clinical application" was held in Brescia, Italy, in March 2009, and aimed to harness an understanding of basic inflammatory mechanisms inherent in human diseases with updated findings regarding biological and therapeutic properties of human placenta-derived cells, with particular emphasis on their potential for treating inflammatory diseases. Finally, steps required to allow their future clinical application according to regulatory aspects including good manufacturing practice (GMP) were also considered. In September 2009, the International Placenta Stem Cell Society (IPLASS) was founded to help strengthen the research network in this field.