Development of tools for in situ hybridization in human embryonic stem cells

Abstract only availableOur laboratory uses human embryonic stem cells (hESC) to understand how embryonic cells differentiate into the cells of the placenta. The long term goal of my research is to develop in situ hybridization methods and use them to determine where genes are being expressed within differentiating hESC colonies. I focused my research on two genes that code for transcription factors, OCT 4 and GATA 2. OCT 4 is required to maintain stem cells in an undifferentiated state. GATA 2 is thought to be one of the key transcription factors that promote stem cell differentiation into syncytiotrophoblast, a placental cell type. The first step in in situ hybridization is the development of a labeled RNA probe. I designed primers to amplify a 214bp portion of the GATA2 gene sequence by PCR, and then cloned the PCR product into E. coli bacteria. I confirmed the presence and orientation of the insert by using restriction digestion and DNA sequencing. I used bacteria already transformed with a 182bp OCT4 probe sequence to create a riboprobe. I grew up the bacteria overnight, lysed them, and purified the plasmid DNA. I linearized the plasmid with a restriction enzyme, purified it, and transcribed the DNA to RNA, incorporating digoxygenin (DIG) labeled nucleotides. In the near future, I will utilize the same method to transcribe the GATA2 probe, and use both RNA probes to perform in situ hybridizations on hESC.Life Sciences Undergraduate Research Opportunity Progra

Offspring sex ratio varies with maternal condition in mice

Abstract only availableField and laboratory animal studies have demonstrated that maternal body condition can directly cause adjustments in offspring sex ratio. A mother's condition is affected by a variety of environmental and endogenous factors, but in particular food availability plays a major determining role. During famine conditions, the females should theoretically bias their offspring's sex ratio towards the sex with the lower reproductive value, e.g. daughters in most species. When resources become more plentiful, however, the dams should bias their offspring towards the sex with the greater reproductive value e.g sons in most species. In our experiment, we have chosen to examine the effects of diet manipulation in NIH Swiss and CF1 mice housed under standard laboratory conditions. This experiment was a follow-up to an initial study performed by this laboratory that indicated mouse mothers fed a very high fat diet (VHF) before conception gave birth to more male offspring. However, female mice fed on the low fat (LF) diet produced more daughters. The aim of our current study is to determine when during gestation the shift in offspring sex ratio is occurring in the females provisioned with the two diets. We are focusing initially on two time periods, at fertilization and post-implantation or day 8 phase of pregnancy. To determine the sex of these early stage embryos, two approaches are being employed: 1) PCR analysis of sex chromosome DNA and 2) Fluorescence in Situ Hybridization (FISH) with X and Y chromosome probes. In the course of our experiments, we have met and are currently working to overcome several problems that have occurred. We are close to optimizing both procedures, and in the next few months, we anticipate having more data and a better understanding of when the shift in offspring sex ratio is occurring in the females fed the LF and VHF diets.Life Sciences Undergraduate Research Opportunity Progra

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

Autoimmunization of Ewes Against Pregnancy-associated Glycoproteins Does Not Interfere with the Establishment and Maintenance of Pregnancy

doi:10.1017/S1751731109004145Pregnancy-associated glycoproteins (PAGs) are a large grouping of placental proteins that belong to the aspartic peptidase gene family. Although useful to detect pregnancy in ruminant species, the function of these molecules is unclear. Several PAGs expressed by trophoblast binucleate cells can enter the maternal circulation, suggesting that they could have a systemic role in altering maternal physiology. The objective of this work was to examine whether these circulating placental antigens were important in pregnancy by actively immunizing ewes against them. PAGs were purified by pepstatin-affinity chromatography and conjugated to the immunogenic protein, keyhole limpet hemocyanin (KLH). Ewes were immunized with PAG-KLH conjugate (n522) or with KLH alone (n59), and bred to intact rams. Blood samples, collected on Day 0 (day of estrus), Day 10, Days 15 to 25 and weekly throughout pregnancy, were analyzed for PAG by an ELISA. On Day 30, pregnancy was confirmed by ultrasound. Ewes immunized against PAG-KLH produced a range of reactive anti-PAG titers, whereas all immunized ewes had high anti-KLH immunoreactivity. PAGs became detectable in the anti-KLH (control) ewes at Day 21.662.2 of pregnancy. Those ewes immunized against PAGs (n57), that had very low immunoreactivity toward PAGs, had measurable PAG by Day 22.961.3, and their PAG serum profiles throughout pregnancy did not differ from the controls. Those exhibiting moderate to high anti-PAG immunoreactivity (n515), had significantly lower PAG concentrations than controls, with antigen not becoming detectable until Day 48.1615.6. The decrease in circulating PAG in the immunized animals did not correlate with changes in pregnancy rates, lamb number or lamb birth weight. These results suggest that while PAGs may play a role in maintaining pregnancy, their major contribution is likely to be at the fetal-maternal interface. Their actions at extra-placental sites are presumably of more secondary importance.This work was supported by NIH Grant HD21896 and the Animal Reproductive Biology Group of the University of Missouri Food for the 21st Century Program

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

Nutritional Skewing of Conceptus Sex in Sheep: Effects of a Maternal Diet Enriched in Rumen-Protected Polyunsaturated Fatty acids (PUFA)

doi:10.1186/1477-7827-6-21Evolutionary theory suggests that in polygynous mammalian species females in better body condition should produce more sons than daughters. Few controlled studies have however tested this hypothesis and controversy exists as to whether body condition score or maternal diet is in fact the determining factor of offspring sex. Here, we examined whether maternal diet, specifically increased n-6 polyunsaturated fatty acid(PUFA) intake, of ewes with a constant body condition score around the time of conception influenced sex ratio.The research was supported by USDA/CSREES/NRI Grant 2001-35203- 10693 (to RMR) and a Life Sciences Molecular Biology Fellowship, University of Missouri (partial salary support for MPG)

US Animal Agriculture : Making the Case for Productivity

This paper documents the tremendous productivity gains that have been made in United States(US)animal agriculture over the past century. Productivity gains will continue to be necessary as global demand for animal protein outpaces world productive capacity. Genetic technologies, with proper oversight and risk assessment, can provide great benefits for years to come.Includes bibliographical reference

Maternal diet and sex of offspring

http://satscience.missouri.edu/satscience_2004/Mike_Roberts_1_28_2006.pdfPresentation with 78 slidesPowerpoint presentation delivered by Curators' Professor in the Department of Animal Science and Biochemistry in the College of Arts and Sciences at the University of Missouri-Columbia, R. Michael Roberts, as part of the Saturday Morning Science lecture series

Characterizing a human embryonic stem cell model of placental development [abstract]

Abstract only availableWhen human embryonic stem cells (hESC) are treated with BMP4, they differentiate into trophoblast cells, the major cell type of the placenta. There are three main types of trophoblasts: syncytio-, cyto-, and extravillous trophoblasts. Here we studied syncytiotrophoblast formation using immunostaining and microarrays to follow changes in expression of genes known to be expressed in placental syncytiotrophoblast and regulated by BMP4 in hESC over time. We also examined the effect of oxygen atmosphere on the differentiation process by comparing cells cultured under high (H) oxygen (20%) and low (L) oxygen (4%) conditions. We narrowed our focus to the protein products of two genes: GATA2, and CGA. GATA 2 is a transcription factor important for syncytiotrophoblast differentiation, while hCG (the product of CGA) is a product of syncytiotrophoblast and a subunit of the pregnancy hormone hCG. Our aim of this project was to determine by immunofluorescence when and where BMP-treated hESC express these two proteins. A time course was performed with two sets of hESC (line H1) cells exposed to BMP4 for one, three and six days under either N or L conditions. At day 1, neither GATA2 nor hCG were expressed, but by day 3 small regions of GATA2-positive cells were evident, some of which were also hCG-positive. By day 6, staining for both proteins was widespread, but the hCG was always present in a subset of GATA2-positive cells. More GATA2/hCG staining was seen under N as compared to L. Microarray analysis showed up-regulation of additional syncytiotrophoblast-associated genes and were consistent with the immunostaining data. We conclude that HES cells treated with BMP express syncytiotrophoblast markers, and that high oxygen accelerates differentiation of hESC

Combinatorial Roles of Protein Kinase A, Ets2 and CBP/p300 in the Transcriptional Control of Interferon-tau Expression in a Trophoblast Cell Line

doi: 10.1210/me.2007-0300In ruminants, conceptus interferon-tau (IFNT) production is necessary for maintenance of pregnancy. We examined the role of protein kinase A (PKA) in regulating IFNT expression through the activation of Ets2 in JAr choriocarcinoma cells. Although overexpression of the catalytic subunit of PKA or the addition of 8-bromo-cAMP had little ability to up-regulate boIFNT1 reporter constructs on their own, coexpression with Ets2 led to a large increase in gene expression. Progressive truncation of reporter constructs indicated that the site of PKA/Ets2 responsiveness lay in a region of the promoter between -126 and -67, which lacks a cAMP response element but contains the functional Ets2-binding site and an activator protein 1 (AP1) site. Specific mutation of the former reduced the PKA/Ets2 effects by more than 98%, whereas mutation of an AP1-binding site adjacent to the Ets2 site or pharmacological inhibition of MAPK kinase 2 led to a doubling of the combined Ets2/PKA effects, suggesting there is antagonism between the Ras/MAPK pathway and the PKA signal transduction pathway. Although Ets2 is not a substrate for PKA, lowering the effective concentrations of the coactivators, cAMP response element-binding protein-binding protein (CBP)/p300, known PKA targets, reduced the ability of PKA to synergize with Ets2, suggesting that PKA effects on IFNT regulation might be mediated through CBP/p300 coactivation, particularly as CBP and Ets2 occupy the proximal promoter region of IFNT in bovine trophoblast CT-1 cells. The up-regulation of IFNT in the elongating bovine conceptus is likely due to the combinatorial effects of PKA, Ets2, and CBP/p300 and triggered via growth factors released from maternal endometrium