derivation-differentiation-canine-ovarian-mesenchymal-stem

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...

University of São Paulo

The bone morphogenetic proteins (BMPs) belong to a unique group of proteins that includes the growth factor TGF-β. 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 São 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.

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

The rock cavies (Kerodon rupestris) are rodents belonging to family Caviidae, with habitat restricted to northeastern Brazil. Current studies have shown that blood cord has stem and hematopoietic cells both with a high regenerative potential, microscopic studies about the description in rodents and other mammals are scarce. The aim of this study was to describe the structural components of the umbilical cord of rock cavies. Rock cavies embryos at different stages of pregnancy were used, three in the first third, five in middle third, and six in the final third of gestation. They were obtained at the Centre for Wild Animals Multiplication of Federal Rural University of Semi-Arid. In rock cavies was observed that the number of vessels involved in the transport of maternal substances was represented by two major arteries, veins and an allantois duct. The umbilical cord was delimited by amniotic epithelium, formed by a layer of squamous cells, connective tissue with characteristic of the mucosal tissue, corresponding to the Wharton jelly (fetal mesenchyme), where elastic fibers were observed and intercellular spaces were arranged in concentric blades. The morphology observed in rock cavies is similar to that described in the literature for other mammals, including man. The rock cavies are well adapted to captivity and are docile, which makes this animal an important model for study within the field of regenerative medicine. The knowledge of the umbilical cord morphology represents a base point for using this animal as a model for culture and cell therapy.

Microscopy of the umbilical cord of rock cavies--Kerodon rupestris Wied, 1820 (Rodenta, Caviidae).

To characterize bone marrow progenitors cells grown in vitro, using native goats from northeastern Brazil as animal model.

Isolation and characterization of mesenchymal progenitors derived from the bone marrow of goats native from northeastern Brazil.

Considering the physiological importance of the pancreas as an endocrine and exocrine organ, this study described the characteristics of the gross and microscopic morphology of this organ using 16 Myrmecophaga tridactyla individuals. The pancreas was located in the left antimere of the body, was pale in colour and exhibited an elongated shape with a central body and lobulated surface. It was positioned in the abdomen, following the curvatura ventriculi major of the stomach, and was attached to the initial portion of the duodenum. The corpus pancreatis was elongated and showed a caudal curvature of 45°. The pancreas exhibited a facies dorsalis (related to the spleen and stomach) and a facies ventralis (related to the renal capsule and intestine). Macroscopically, a craniodorsal, medial, and caudoventral regions were identified, in addition to the left lobe. Structurally, the organ exhibited two distinct parts: the first had exocrine characteristics, consisting of acini and ducts; the second, which was the endocrine portion, consisted of the pancreatic islets, which were located in the medial, caudoventral and left lobe regions. Ultrastructural analysis identified secretory vesicles containing zymogen granules, mitochondria, Golgi apparatus and rough endoplasmic reticulum in pancreatic centroacinar cells. Morphological data on the anatomy of members of the Xenarthra have revealed important peculiarities of several organs and systems, adding great biological value to the representatives of this group. In addition, these studies significantly contribute not only to knowledge of the biology, taxonomy and, consequently, preservation of these animals but also to the discovery of new experimental models. Anat Rec, 300:1104-1113, 2017. © 2016 Wiley Periodicals, Inc.

Trend Toward Individualization of the Endocrine and Exocrine Portions of the Giant Anteater Pancreas (Myrmecophaga Tridactyla, Xenarthra).

Stem cells are capable of unlimited self-renewal and are able to remain undifferentiated for extended periods of time prior to their differentiation into specific cell lineages. Because of the issues (ethical and religious) involved in the use of embryonic stem cells and the limited plasticity of adult stem cells, an alternative cell source could be foetal stem cells derived from extra-embryonic tissue, which are highly proliferative, grow in vitro and possess interesting immunogenic characteristics. As a result, the amniotic membrane of several species has been studied as an important new source of stem cells.

Phenotype and multipotency of rabbit (Oryctolagus cuniculus) amniotic stem cells.

Considering the limited number of available lung donors, lung bioengineering using whole lung scaffolds has been proposed as an alternative approach to obtain lungs suitable for transplantation. However, some decellularization protocols can cause alterations on the structure, composition, or mechanical properties of the lung extracellular matrix. Therefore, the aim of this study was to compare the acellular lung mechanical properties when using two different routes through the trachea and pulmonary artery for the decellularization process. This study was performed by using the lungs excised from 30 healthy male C57BL/6 mice, which were divided into 3 groups: tracheal decellularization (TDG), perfusion decellularization (PDG), and control groups (CG). Both decellularized groups were subjected to decellularization protocol with a solution of 1% sodium dodecyl sulfate. The behaviour of mechanical properties of the acellular lungs was measured after decellularization process. Static (Est) and dynamic (Edyn) elastances were obtained by the end-inspiratory occlusion method. TDG and PDG showed reduced Est and Edyn elastances after lung decellularization. Scanning electron microscopy showed no structural changes after lung decellularization of the TDG and PDG. In conclusion, was demonstrated that there is no significant difference in the behaviour of mechanical properties and extracellular matrix of the decellularized lungs by using two different routes through the trachea and pulmonary artery.

Effects of two different decellularization routes on the mechanical properties of decellularized lungs.

The incidence of diabetes mellitus in dogs is increasing in recent years, mainly because of genetic and/or environmental factors, including endocrine disorders (like in humans); failure of suitable control of blood sugar levels, which triggers hyperglycemia; glycosuria and weight loss, which demands the development of innovative treatments to cure or treat this complex disease in dogs. The present study established for the first time a protocol to obtain and characterize cells derived from pancreas of canine fetuses. Those fetuses do not have a defined breed and were at the final stage of gestation. The protocol aims to provide morphological data to enable future applications of these cells for therapeutic approaches. In cell culture, pancreatic cells showed a fibroblast-like appearance with a mono-layered growth pattern and were not tumorigenic. They exhibited a positive expression for the pluripotent proliferation markers NANOG and PCNA and expressed PDX1, a transcription factor that is important for activation of the insulin gene promoter. In addition, Tyrosine Hydroxylase-positive (TH+) sympathetic nerve fibers were identified. Histologically, the pancreatic epithelium was developed, pancreatic glands in the fetuses were like those in the parenchyma of postconception dogs and pancreatic islets were unevenly distributed and organized in small clusters along the glands close to the vasculature. Staining with dithizone indicated the presence of insulin in the cells. A large number of beta cells were confirmed by immunofluorescence. In conclusion, the canine fetal pancreas cells could be an alternative and adequate source of cell lineages for stem cell therapies for diabetes treatment. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc.

Isolation and Characterization of Pancreatic Canine Fetal Cells at the Final Stage of Gestation.

Breast cancer is the most common cancer in women, but the incidence of mammary carcinoma in female dogs is even higher than in humans. These two tumors have similarities that can be seen by its biological behavior, molecular genetic alterations, and histology. This suggest that female dogs can be an excellent model for preclinical oncological studies. And the mammary carcinoma most frequently found in this species is the tubular and solid carcinomas. The extracellular matrix (ECM) has an important role in the progression of these tumors. Because of that we proposed to evaluate the ECM components of these carcinomas through histology with specific stains such as Masson's Trichrome, Picrosirius Red and the technique of scanning electron microscopy. With that, we found the presence of collagen fibers in the tubular carcinoma and around its parenchyma. On the other hand, the solid carcinoma presented collagen fibers throughout the parenchyma and around each tumor cell. With the transmission electron microscopy, we observed the presence of mitochondrias and rough endoplasmic reticulum in both tumors. And finally, we evaluated the expression of proteins through the immunohistochemistry, in which we found a high expression of VEGF, PCNA, CK-18 and vimentin in solid carcinoma, and a positive mark in the tubular and solid carcinoma for collagen I, III and fibronectin. Thus, we demonstrated some differences in the ECM of these mammary carcinomas, allowing a better understanding of its histological characteristics, and these data may contribute to future studies about therapies focused on tumors ECM.

Evaluation of immunohistopathological profile of tubular and solid canine mammary carcinomas.

The reason why shearing ewes in mid-pregnancy does increase the lamb birth weight is not completely clears. Therefore, we focused on the analyses of the deposition of glycogen in different fetal tissues to investigate this issue. Thirteen pregnant Australian Merino ewes, raised in native pasture, were separated in two groups. One group (n = 7) was shorn (SE) at 70 days of pregnancy, whereas another group (n = 6) remained unshorn (NSE). Cesarean section was conducted in all the ewes at near parturition, when placenta and fetuses sampling were collected. Placenta, liver and muscle samples were fixed and stained with glycoprotein-reactive acid-Schiff acid for analysis under light microscopy. The quantification of these glycoproteins was performed with the support of a program that analyzes the measurement of the intensity of staining by field. Five random fields from each sample were used, where statistical analyzes was used as normal test T. Among the analyzed regions, the deposition of glycoprotein between SE and NSE groups was statistically different in the hepatic portal vein (54,499.23 µm  in SE and 34,830.73 µm in NSE) and in the total muscle area of the sample fragment (41,128, 7 µm and 31,942.7 µm , respectively; P < 0.05). We conclude that shearing ewes at the 70th day of gestation lead to accumulation of glycoproteins in the liver and muscle of fetuses, which may be responsible for the increase in birth weights in that group.

Maria Angélica Miglino

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University of São Paulo

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

FMVZ