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 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.