An analysis of aspartic peptidases expressed by trophoblasts and placenta of even-toed ungulates

Title from PDF of title page (University of Missouri--Columbia, viewed on February 23, 2010).The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file.Dissertation advisor: Dr. Jonathan A. Green.VitaPh.D. University of Missouri--Columbia 2008.The Pregnancy Associated Glycoproteins (PAGs) represent a multigene family of trophoblast expressed proteins, found exclusively in the placenta of even-toed mammals such as ruminants (cattle, sheep), pig, etc. In ruminants, the PAGs can be classified into ancient and modern PAGs based on their coding sequence. In addition, there are also differences in purported enzymatic activity as well as transcriptional regulation of expression. Many of the modern PAGs have accumulated mutations in and around the catalytic center, and some of those that incurred mutations in the two catalytic aspartates are predicted to be proteolytically inactive. In contrast, most of the ancient PAGs of ruminants and swine, have all the hallmarks of typical aspartic peptidases (APs). From the analysis of cattle genome, we found that there are 18 distinct PAG genes and 14 pseudogenes. Based on our preliminary analysis of the proximal promoter regions [500 base pairs (bp) upstream of the translational start point] of PAG genes, we found that there are pockets of conserved transcription factor binding sites that are different between ancient and modern PAGs. These differences likely influence the observed differences in expression between ancient and modern boPAGs. We gathered evidence by Real-time PCR and global analysis of expressed ESTs that confirm that, boPAG-2 is the most abundant of all boPAGs. We identified boPAG-2 and its closest paralog boPAG-12, as well as poPAG-2 the ancient PAG found in pigs, as the candidates for investigation of proteolytic activity. From our experiments we found that, boPAGs -2 and -12 and poPAG-2 are proteases with optimal activity under acidic pH conditions. We also illustrated differences in proteolytic activity towards substrates, and in their relative affinity towards an AP inhibitor (pepstatin A). We found that, in comparison to the two bovine paralogs, boPAGs -2 and -12, poPAG-2 was found to be a more robust enzyme. Finally, we demonstrated that APs secreted by embryos such as PAGs can be objectively measured in the medium conditioned by the culture of porcine embryos either individually or in pools for variable lengths of time in acidic conditions. We also observed that such activity seemed to correlate with stage and quality of embryos (assessed morphologically) in vitro. We, therefore, believe that this proteolytic activity potentially could serve as a marker for developmental competence of the embryos.Includes bibliographical references

Derivation and characterization of LIF and FGF2 dependent Porcine Induced Pluripotent Stem Cells [abstract]

Comparative Medicine - OneHealth and Comparative Medicine Poster SessionAuthentic embryonic stem cells (ESC) have probably never been derived from the inner cell mass (ICM) of pig, despite over 25 years of effort. Recently, several groups, including ours, have reported derivation of induced pluripotent stem cells (iPSC) from swine by reprogramming somatic cells with a combination of four factors (OCT4, SOX2, KLF4, C-MYC) delivered by retroviral transduction. The piPSC resembled FGF2- dependent human (h) ESC and are likely to advance swine as a model in biomedical research, since grafts could potentially be matched to the animal that donated the cells for re-programming. The dependence of piPSC on FGF2 also draws parallels to murine pluripotent stem cells derived from advanced epiblast, so-called 'epiblast stem cells'. Indeed, an emerging concept is that there are two kinds of ESC, one dependent on FGF2 and not competent to contribute to germ-line chimeras, the other upon LIF and germ-line competent. The objective of our recent investigations has been to develop LIF- dependent piPSC by using the same reprogramming factors but selecting the colonies on a modified LIF- medium supplemented with two kinase inhibitors, CHIR99021, which inhibits GSK-3beta, and kenpaullone, which inhibits both GSK-3beta and CDK1. The LIF-dependent piPSC, derived here from outgrowths of umbilical cord mesenchyme, expressed markers consistent with pluripotency and bore a striking resemblance to ICM-derived murine ESC in colony morphology, culture characteristics, and short cell cycle time. Currently, the ability of LIF-piPSC to give rise to teratoma and chimeras is under investigation. Supported by Missouri Life Sciences Board Grant 00022147 and NIH grant HD21896

Porcine induced pluripotent stem cells (piPSC) for expanding the use of swine in biomedical research

Comparative Medicine - OneHealth and Comparative Medicine Poster SessionOur goal is to create porcine pluripotent stem cells, i.e. ones capable of differentiating into all cell types of the body that can expand the use of swine as a biomedical model for studying human disease. It is well established that mouse embryonic stem cells (ESC) are an excellent source of material for successful cloning and for incorporation into chimeras. However, the establishment of porcine ESC from the embryos has proven to be elusive. There has been a similar lack of success with other ungulate species. Establishing a technology for deriving induced pluripotent stem cells (iPSC) from farm animals will allow the gene knock-in/knock-out methods that have revolutionized mouse genetics to be applied to farm species. Importantly pig is a potentially useful model for studying human pathologies due to similarities in organ size, immunology and whole animal physiology between the two species. If the safety and efficacy of stem cell transplantation is to be tested in an animal model before being applied to humans, the pig would likely be a species of choice. The ability to derive porcine (p) iPSC lines from a particular outbred animal and conduct tissue transplantation on the same pig later and follow the success of the transplant over the course of months or even years would be a particularly valuable advance. Additionally the ability to provide piPSC from animals with valuable traits would provide a permanent source of cells for clonal propagation that would likely avoid the inefficiencies and problems arising from somatic cell nuclear transfer (SCNT), where the vast majority of cloned offspring die or are developmentally abnormal. We have created piPSC from embryonic fibroblasts and umbilical cord mesenchyme by a similar strategy used for the mouse and human, namely ectopically expressing reprogramming genes in somatic cells. The piPSC resemble human ESC, express the typical gene and surface antigen markers of ESC, proliferate continuously in culture, possess high telomerase activity, form embryoid bodies, and differentiate along the three main germ line lineages. Our aim is to demonstrate that piPSC can be directed to differentiate along defined lineages, specifically towards neuronal tissue, hematopoietic lineages and various mesoderm derivatives including cardiomyocytes by using protocols based on those used successfully with human and murine ESC. These experiments will allow such cells to be used for tissue grafts that are matched genetically to recipients and tested for their safety in transplantation. We shall also establish parameters for routine gene targeting in piPSC, with the ultimate goal of creating genetic models for human diseases where mouse models are inappropriate. In summary, the piPSC lines developed will have enormous utility for exploiting the pig as a model in human pre-clinical applications. Supported by Missouri Life Sciences Board Grant 00022147 and NIH grant HD2189