Doctor of Philosophy / Department of Animal Sciences and Industry / Duane L. Davis / Intrauterine growth restriction (IUGR) affects all mammals. In the swine industry IUGR pigs result from intrauterine crowding. Prenatal programming in IUGR pigs has substantial effects on myogenesis and adipogenesis. Prenatal programming due to IUGR is also a problem in humans and long-term effects on adipogenesis are well established for small for gestational age (SGA) babies. Mesenchymal stem cells (MSCs) are the precursors for adipocytes. The umbilical cord contains a population of MSCs in Wharton’s jelly (WJ) and they can be harvested postnatally without ethical issues. Therefore, WJMSCs are proposed as models for studying prenatal programming of adipogenesis. We selected genes from studies of adipogenesis in humans and other species and examined their expression in pig WJ. We assigned pigs within litter as High, Medium, or Low birth weight and evaluated these categories for expression of Cox1, Cox2, EGR1, PPARɣ1, PPARɣ2, and Pref1. Differences due to size classification within litter were limited but there were correlations between weaning weight and delta cycle threshold (ΔCt) for EGR1 (r = 0.28; P < 0.009), PPARɣ1 (r = 0.29; P < 0.007), and PPARɣ2 (r = 0.30; P < 0.005). This may be consistent with the reports for SGA babies where EGR1 is upregulated by prenatal growth restriction. To gain insight into when during pregnancy IUGR affects WJ cells we collected umbilical cords at d 60 and d 95. In d 60 umbilical cords, small fetuses had increased (P = 0.06) Cox1 gene expression. We tested the ability of d 60 WJ cells to undergo adipogenic differentiation using standard protocols and a cycling protocol that exposed the cells to adipogenic differentiation conditions interposed with a rest phase with high insulin. It has been reported that the cycling protocol revealed increased glucose uptake in WJ cells from human SGA babies. We found that d 60 WJ cells did not show adipogenic differentiation in any of the protocols tested however glucose uptake correlated negatively with birth weight at Cycle 0 (P < 0.02; r = 0.61). In summary, pig WJ cells reveal some effects of IUGR but they appear to differ from the relationship demonstrated reported for human SGA babies. A new finding was that at midgestation pig WJ cells do not appear to be competent to complete adipogenesis. We also studied nursing managements to improve outcomes for IUGR pigs. Colostrum intake may be a problem, particularly for light weight pigs and those born later during farrowing. Split suckling is the removal of some pigs to allow others unrestricted nursing access. We temporarily removed the six heaviest pigs and this treatment increased gain and weight by d 7 of age. Colostrum intake was highest for the high birth weight pigs. When we temporarily removed the first half of the litter, colostrum intake was increased for the second half of litter born and the difference in immunocrit was reduced between the two litter halves.

Doctor of Philosophy / Department of Anatomy and Physiology / Mark L. Weiss / Graft Versus Host Disease (GVHD) is the major complication following hematopoietic stem cell transplantation. GVHD is activated by immunocompetent T cells presented in the donor grafted tissue. Due to the increased use of bone marrow transplantation to treat diverse malignancies, the incidence of GVHD has shown a notable increase. Depending of the degree of immunological mismatch between donor and host, 50-70% of patients develop GVHD after allogeneic Bone Marrow Transplantation (BMT). Once GVHD develops, mortality reaches up to 50% in humans. Several studies using Mesenchymal Stromal Cells (MSCs) to prevent and treat GVHD have produced controversial results. It is thought that distinct MSCs sources used in those studies might be an important factor that produces different outcomes. For cellular therapy, the most attractive characteristics of MSCs are their reduced immunogenic potential, and their abilities to modulate immune responses. This dissertation addressed the hypothesis that Wharton’s jelly cells (WJCs) would prevent the pathology and death associated with GVHD after BMT. To accomplish this, I created a clinically relevant model of GVHD by transplanting allogeneic bone marrow across minor histocompatibility antigen (HA) barriers in the rat. To enhance alloreactive T-cell stimulation, bone marrow (BM) was co-administered with a fraction of CD8[superscript]+ cells magnetically selected from spleen to induce GVHD. Bone marrow tissue was isolated from a donor rat Fischer 344 (F344, RT1lv) and transplanted into lethally irradiated (10 Gray) Lewis rat (LEW, RT1l). Once GVHD was induced, MSCs derived from umbilical cord WJCs were either co-transplanted at day 0 with bone marrow, or given on day 2 post-BMT intravenously. The prophylactic potential of WJCs in an in vivo GVHD model was assessed as survival time, clinical symptomatology occurrence, and histopathology injuries in target tissues. Results indicate that while co-administration of WJCs with hematopoietic cells on day 0 failed to alleviate GVHD associated symptomatology and mortality. WJCs administered on day 2 post-induction ameliorated GVHD-associated symptomatology, improved engraftment and survival.

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.