Master of Science / Department of Anatomy & Physiology / Masaaki Tamura / Rat umbilical cord matrix stem cells (UCMSC) have been shown to exhibit a remarkable ability to control rat mammary adenocarcinoma (Mat B III) cell proliferation both in vivo and in vitro. To study the underlying mechanisms and genes involved in Mat B III growth attenuation, total RNA was extracted from the naïve rat UCMSC alone and those co-cultured with Mat B III in Transwell culture dishes. Gene expression profiles of naive rat UCMSC alone and those cocultured with Mat B III cells were investigated by microarray analysis using an Illumina RatRef- 12 Expression BeadChip. The comparison of gene expression profiles between untreated and cocultured rat UCMSC identified five up-regulated candidate genes (follistatin (FST), sulfatase1 (SULF-1), glucose phosphate isomerase (GPI), HtrA serine peptidase (HTRA1), and adipocyte differentiation-related protein (ADRP)) and two down-regulated candidate genes (transforming growth factor, beta-induced, 68kDa (TGFβI) and podoplanin (PDPN)) based upon the following screening criteria: 1) expression of the candidate genes should show at least a 1.5 fold change in rat UCMSC co-cultured with Mat B III cells; 2) candidate genes encode secretory proteins; and 3) they encode cell growth-related proteins. Following confirmation of gene expression by real time-PCR, ADRP, SULF-1 and GPI were selected for further analysis. Addition of specific neutralizing antibodies against these three gene products individually in co-cultures of 1:20 rat UCMSC:Mat B III cells significantly increased cell proliferation, implying that these gene products are produced under the co-cultured condition and functionally attenuate cell growth. Immunoprecipitation followed by Western blot analysis demonstrated that these proteins are indeed secreted into the culture medium. Individual over-expression of these three genes in rat UCMSC significantly enhanced UCMSC-dependent inhibition of cell proliferation in co-culture. These results suggest that ADRP, SULF-1 and GPI act as tumor suppressor genes, and these genes might be involved in rat UCMSC-dependent growth attenuation of rat mammary tumors.

Master of Science / Department of Animal Sciences and Industry / Duane L. Davis / The three transcription factors, Nanog, Oct-4 and Sox-2, are central regulators of pluripotency in embryonic stem cells. Porcine umbilical cord (PUC) matrix stem cells also express these transcription factors. Wharton’s jelly is composed of an extracellular matrix high in hyaluronic acid and various collagens and serves as a reservoir for several growth factors and cytokines. We expect that Wharton’s jelly includes a stem cell niche that provides a microenvironment that maintains and supports the stem-cell characteristics of PUCs. The mechanisms by which the PUCs remain primitive within the Wharton’s jelly are unknown. We developed methods for producing an extracellular matrix product extracted from porcine Wharton’s jelly that we named Pormatrix (PMX). When PMX is incubated at 37[degrees]C, it becomes a matrical gel that provides a matrix allowing PUC attachment and growth. Concentrating the protein in PMX by filtration provides a low molecular weight by-product which we refer to as flow through (FT). In Experiment 1, PUCs were seeded on Pormatrix, Matrigel or plastic substrates in the presence or absence of FT. PUCs cultured on Matrigel, Matrigel+FT, Plastic+FT and PMX had higher expression of Nanog compared to PUCs cultured on PMX+FT (P-value <0.05). In Experiment 2, the PMX and Matrigel were diluted to low protein concentrations (1.2-1.5 mg/ml protein) so that gelling did not occur. Adding FT to PMX, Matrigel and plastic increased gene expression of Nanog 2.78 fold compared to treatments without FT (P =0.10). Sox-2 expression was increased by adding FT to Matrigel but adding FT to the other matrix proteins had no effect resulting in a tendency for a matrix*FT interaction(P=0.10). The transcription factor Oct-4 remained unchanged regardless of treatment. To evaluate the effects of in vitro maintenance on Nanog, Oct-4 and Sox-2 we measured the relative gene expression in PUCs over the first six passages in vitro. Nanog, Oct-4 and Sox-2 did not differ over these passages. This may indicate that during the first six passages in vitro, PUCs remain relatively primitive. In summary, we prepared an extract from Wharton’s jelly from porcine umbilical cords. The extract supported PUC attachment and growth and appeared to regulate gene expression. Perhaps with further investigation the interactions of PUCs with their in vivo environment can be elucidated.

Doctor of Philosophy / Department of Animal Sciences and Industry / Duane Davis / Umbilical cords matrix stem cells (UCs) have been characterized most thoroughly in humans (HUCs) and are considered to have great promise for regenerative medicine and cell-based therapy. Although UCs were first identified in pigs the description of porcine UCs (PUCs) is limited. Here we reported some standard mesenchymal stem cell characteristics for PUCs. Development of knowledge about PUCs is useful because the pig is a valuable biomedical model for humans and the species is an important human food source. PUCs were isolated from Wharton’s jelly using an explant technique. They attached on the plastic and showed fibroblast-like morphology. Immunophenotype analysis showed they are positive for CD44, CD90 and CD105 and negative for CD31, CD45 and SLA-DR. Under specific in vitro conditions, PUCs were differentiated to adipocytes, chondrocytes and osteocytes. The growth curve of PUCs exhibited a lag phase, log phase and doubling time of 24, 60 and 13.8 hour respectively. Engraftment potential of allogeneic PUCs administered orally and intraperitoneally (IP) was evaluated. Newborn, 1-day, 1-week, 2-week and 3-week old pigs were administered a dose of fluorescently labeled PUCs (1.1x107 cells/kg body weight) and their tissue incorporation were evaluated using confocal microscopy with confirmation by PCR to detect SRY gene, the Y-chromosome gene of male PUCs in female recipients. One week after PUCs administration, they were found mostly in the gastrointestinal tract and abdominal organs after either oral or intraperitoneal transplantation. The intestinal mucosa layer around the base of villi and intestinal crypts was the main location. PUCs were also detected in thoracic organs, muscle and bone marrow. Additionally, PKH26-labeled fibroblasts labeled were detected in recipient intestine 1 week after IP injection. Donor cells were not found in blood at one week post transplantation. When recipients were sacrificed at 6 h after IP injection PKH26-labeled PUCs were found mostly in omentum and diaphragm by PCR. It is likely these are the primary sites for donor cells in the peritoneal cavity to enter the circulation. Fluorescent in situ hybridization (FISH), using an SRY probe and PCR, demonstrated the PUCs isolated from recipient intestines by enzymatic digestion. Therefore, transplanted PUCs were recovered from the intestinal mucosa and were viable and able to proliferate in vitro.

Master of Science / Department of Anatomy and Physiology / Masaaki Tamura / Previous studies have shown that both human and rat umbilical cord matrix mesenchymal stem cells (UCMSC) possess the ability to control the growth of breast carcinoma cells. Comparative analysis of two types of UCMSC suggest that rat UCMSC-dependent growth regulation is significantly stronger than that of human UCMSC. Accordingly, the present study was designed to clarify their different tumoricidal abilities by analyzing gene expression profiles in two types of UCMSC. Gene expression profiles were studied by microarray analysis using Illumina HumanRef-8-V2 and RatRef-12 BeadChip for the respective UCMSC. The gene expression profiles were compared to untreated naïve UCMSC and those co-cultured with species-matched breast carcinoma cells; human UCMSC vs. MDA-231 human carcinoma cells and rat UCMSC vs. Mat B III rat carcinoma cells. The following selection criteria were used for the screening of candidate genes associated with UCMSC-dependent tumoricidal ability; 1) gene expression difference should be at least 1.5 fold between naive UCMSC and those co-cultured with breast carcinoma cells; 2) they must encode secretory proteins and 3) cell growth regulation-related proteins. These analyses screened 17 common genes from human and rat UCMSC. The comparison between the two sets of gene expression profiles identified that two tumor suppressor genes, adipose-differentiation related protein (ADRP) and follistatin (FST), were specifically up-regulated in rat UCMSC, but down-regulated in human UCMSC when they were co-cultured with the corresponding species’ breast carcinoma cells. The suppression of either protein by the addition of a specific neutralizing antibody in co-culture of rat UCMSC with Mat B III cells significantly abrogated UCMSC ability to attenuate the growth of carcinoma cells. Over-expression of both genes by adenovirus vector in human UCMSC enhanced their 4 ability to suppress the growth of MDA-231 cells. In the breast carcinoma lung metastasis model generated with MDA-231 cells, systemic treatment with FST-over-expressing human UCMSC significantly attenuated the tumor burden. These results suggest that both ADRP and FST may play important roles in exhibiting stronger tumoricidal ability in rat UCMSC than human UCMSC and imply that human UCMSC can be transformed into stronger tumoricidal cells by enhancing tumor suppressor gene expression.

Master of Science / Department of Anatomy and Physiology / Mark L. Weiss / Wharton's jelly is a non-controversial source of mesenchymal stromal cells. Isolation of the cells is non-invasive and painless. The cells have been shown to have a wide array of therapeutic applications. They have improved symptoms when transplanted in a variety of animal disease models, can be used in tissue engineering applications to grow living tissue ex vivo for transplantation, and can be used as drug delivery vehicles in cancer therapy. The cells have also been shown to be non-immunogenic and immune suppressive. This thesis focuses on optimizing isolation protocols, culture protocols, cryopreservation, and characterization of cells in different growth conditions. Results from the experiments indicate that isolation of cells by enzyme digestion yields cells consistently, a freezing mixture containing 90% FBS and 10% DMSO confers maximum viability, and the expression of mesenchymal stromal cell consensus markers does not change with passage and cryopreservation. The results of the experiments also show that cells grow at a higher rate in 5% oxygen culture conditions compared to 21% oxygen culture conditions, serum does not have an effect on growth of the cells, serum and oxygen do not have effects on the expression of mesenchymal stromal cell consensus markers and the cells are stable without nuclear abnormalities when grown in 5% oxygen and serum free conditions for six passages after first establishing in serum conditions.

Master of Science / Department of Animal Sciences and Industry / Duane L. Davis / Intestinal absorption of colostral leukocytes (CL), peripheral blood mononuclear cells (PBMC), and porcine umbilical cord matrix stem cells (PUC) was analyzed in neonatal pigs. Maternal CL have previously been demonstrated in pigs, and maternal PBMC have been observed in calves to enter neonatal circulation after ingestion. PUC are primitive stem cells that are easily isolated from Wharton's jelly of the porcine umbilical cord. These cells do not have an immunogenic effect on the host upon initial transplantation. The general characteristics of PUC may allow them to serve as a delivery system to the neonate. Cellular migration through the duodenum, jejunum, and ileum was assessed using confocal microscopy. In vitro experiments utilized an organ explant culture system to determine the trafficking of labeled cells. Small-intestine tissue was collected from stillborn and sacrificed neonates. All three cell types (CL, PBMC, and PUC) were detected below the luminal surface, after 72 h of culture with media, and regardless of whether explants were from stillborns or live-born pigs. In vivo trafficking was assessed using neonatal pigs that were fed PBMC isolated from their mother or PUC from an unrelated pig. The effect of prior exposure to 25% acellular colostrum (AC) in medium was evaluated for both cell types. Piglets were euthanized 8 h or 24 h post feeding and sections of the small intestine collected. Both PBMC and PUC were found in all intestinal samples. Exposure to AC had no detected effect on the ability of either cell type to attach and migrate into the tissue. Labeled PUC were detected on the surface of the epithelium and in the lamina propria 8 h post treatment. PBMC were observed on the surface of the epithelium, in the lamina propria, and superficial submucosa 8 h following ingestion. In neonates sacrificed 24 h post treatment, both PUC and PBMC were observed on the surface of the epithelium, in the lamina propria, superficial submucosa, and deep submucosa of the small intestine. PUC and PBMC were noted at the apex, intermediate between the apex and the base, or at the base of the villus.

Les affections articulaires touchant le cartilage, telles que les lésions focales et l’arthrose, correspondent aux principales causes de baisse de performance et d’arrêt prématuré de la carrière sportive du cheval. Ainsi, le traitement des affections du cartilage représente un enjeu vétérinaire majeur dans le monde équin, du fait des importantes pertes financières qu’elles occasionnent à la filière. Les faibles capacités de réparation intrinsèque du cartilage, ainsi que l’absence de thérapie à long terme des dommages cartilagineux, nécessitent le recours à des thérapies de nouvelles générations telle que l’ingénierie tissulaire du cartilage. Dans ce cadre, notre étude s’est attachée à comparer différents types cellulaires pour la génération de cartilage in vitro, afin d’envisager une implantation pour traiter les atteintes cartilagineuses chez le cheval. Une technique initialement développée chez l’Homme, la transplantation de chondrocytes autologues, représente toujours un « gold standard » en ingénierie tissulaire du cartilage. Dans ce travail de thèse, après avoir développé une nouvelle génération de substitut cartilagineux de haute qualité biologique, à partir de chondrocytes articulaires équins, des limites techniques et biologiques inhérentes au type cellulaire persistent. Ainsi, nos travaux se sont tournés vers la recherche de types cellulaires alternatifs. Les cellules souches/stromales mésenchymateuses (CSM) néonatales issues de cordon ombilical telles que les CSM de sang placentaire (CSM-SPL) et les CSM de gelée de Wharton (CSM-GW) pourraient représenter un avantage thérapeutique du fait de leur isolement non-invasif, de leur forte prolifération cellulaire et de leur capacité de différenciation en chondrocyte. Il est néanmoins indispensable de définir le meilleur candidat thérapeutique, parmi ces deux sources cellulaires, pour l’obtention d’un substitut cartilagineux de qualité biologique optimale. Ces résultats de thèse ont montré d’importantes différences dans le processus de chondrogenèse de ces deux sources de CSM néonatales et plaident en faveur de l’utilisation des CSM-SPL dans le cadre d’une stratégie thérapeutique d’ingénierie tissulaire du cartilage équin. Ces travaux ont permis une meilleure compréhension de la biologie du chondrocyte et des CSM. De surcroît, ces travaux permettent d’envisager de futurs essais cliniques chez le cheval, afin de traiter les affections articulaires de ce modèle gros animal. / Articular cartilage disorders, such as focal defects and osteoarthritis, are the main causes of decreased performance or early retirement of sport- and racehorses. Thus, cartilage disorders represent a major veterinary issue in the equine industry, due to significant financial losses. Poor intrinsic cartilage repair properties and the absence of long- term therapy for cartilage defects lead to the development and use of new generation therapies such as autologous chondrocytes implantation. In this context, our study aimed to compare different cell types for the in vitro cartilage generation, in order to implant the biological substitute to treat cartilage defects in the horse. A therapeutic strategy initially developed in human medicine, the autologous chondrocytes transplantation, always represents a "gold standard" in cartilage tissue engineering. In the present study, after developing a new generation of cartilaginous substitute of high biological quality, composed of equine articular chondrocytes, technical and biological limits inherent to the cell type persist. Thus, we have used alternative cell types such as neonatal mesenchymal stem/stromal cells (MSCs) from umbilical cord, such as umbilical cord blood MSC (UCB-MSCs) and umbilical cord matrix or Wharton jelly MSCs (UCM- MSCs). These MSCs sources could represent a therapeutic advantage due to their non-invasive isolation, their high cell proliferation and their ability to differentiate into chondrocytes. Nevertheless, it is essential to define the best therapeutic candidate between these two MSCs sources, to obtain an optimal quality for the neocartilaginous substitute. Our data highlighted important differences in the chondrogenesis process of these two neonatal MSCs sources, allowing us to consider UCB-MSCs as the best therapeutic candidate for equine cartilage tissue engineering. This work allows a better understanding of the chondrocyte and MSCs biology. Moreover, this work leads the way to setting-up future clinical trials in the horse, in order to treat articular defects of this large animal model.