Gender dependent survival of allogeneic trophoblast stem cells

Pregnancy succeeds because the fetal allograft survives in the presence of a fully functional maternal immune system. The placenta, especially its trophoblast, provides the initial barrier between the maternal and fetal environment and, due to their location, trophoblast cells could be expected to be immune-privileged. Yet in the ectopic sites tested thus far, trophoblast stem cell transplants have failed to show noticeable immune privilege and appear to lack physiological support. However in this study, portal vein injected green fluorescent protein-labeled trophoblast stem cells were able to survive for several months in the livers of allogeneic female (14/14), but not male (0/4), mice. Gonadectomy experiments revealed that this gender-dependent survival does not require the presence of ovarian hormones (4/4) but the absence of testicular factors (5/5). In contrast, similarly labeled allogeneic embryonic stem cells were reliably rejected (11/11); these same embryonic stem cells survived when mixed with unlabeled trophoblast stem cells (13/13). The protective effect offered by the trophoblast stem cells did not require any immunological similarity with the co-injected embryonic stem cells. Neither the trophoblast stem cells nor the co-injected embryonic stem cells gave rise to tumors during the study period. Thus, this study demonstrates that, provided a suitable location and hormonal context, ectopic trophoblast stem cells may exhibit and confer immune privilege. These findings suggest applications in cell and gene therapy as well as provide a new model for studying trophoblast physiology and immunology

Gender dependent survival of allogeneic trophoblast stem cells

Pregnancy succeeds because the fetal allograft survives in the presence of a fully functional maternal immune system. The placenta, especially its trophoblast, provides the initial barrier between the maternal and fetal environment and, due to their location, trophoblast cells could be expected to be immune-privileged. Yet in the ectopic sites tested thus far, trophoblast stem cell transplants have failed to show noticeable immune privilege and appear to lack physiological support. However in this study, portal vein injected green fluorescent protein-labeled trophoblast stem cells were able to survive for several months in the livers of allogeneic female (14/14), but not male (0/4), mice. Gonadectomy experiments revealed that this gender-dependent survival does not require the presence of ovarian hormones (4/4) but the absence of testicular factors (5/5). In contrast, similarly labeled allogeneic embryonic stem cells were reliably rejected (11/11); these same embryonic stem cells survived when mixed with unlabeled trophoblast stem cells (13/13). The protective effect offered by the trophoblast stem cells did not require any immunological similarity with the co-injected embryonic stem cells. Neither the trophoblast stem cells nor the co-injected embryonic stem cells gave rise to tumors during the study period. Thus, this study demonstrates that, provided a suitable location and hormonal context, ectopic trophoblast stem cells may exhibit and confer immune privilege. These findings suggest applications in cell and gene therapy as well as provide a new model for studying trophoblast physiology and immunology

Gender-Dependent Survival of Allogeneic Trophoblast Stem Cells in Liver

In view of the well-known phenomenon of trophoblast immune privilege, trophoblast stem cells (TSCs) might be expected to be immune privileged, which could be of interest for cell or gene therapies. Yet in the ectopic sites tested so far, TSC transplants fail to show noticeable immune privilege and seem to lack physiological support. However, we show here that after portal venous injection, green fluorescent protein (GFP)-labeled TSCs survive for several months in the livers of allogeneic female but not male mice. Gonadectomy experiments revealed that this survival does not require the presence of ovarian hormones but does require the absence of testicular factors. By contrast, GFP-labeled allogeneic embryonic stem cells (ESCs) are reliably rejected; however, these same ESCs survive when mixed with unlabeled TSCs. The protective effect does not require immunological compatibility between ESCs and TSCs. Tumors were not observed in animals with either successfully engrafted TSCs or coinjected ESCs. We conclude that in a suitable hormonal context and location, ectopic TSCs can exhibit and confer immune privilege. These findings suggest applications in cell and gene therapy as well as a new model for studying trophoblast immunology and physiology

Isolation of Oct4-Expressing Extraembryonic Endoderm Precursor Cell Lines

BACKGROUND:The extraembryonic endoderm (ExEn) defines the yolk sac, a set of membranes that provide essential support for mammalian embryos. Recent findings suggest that the committed ExEn precursor is present already in the embryonic Inner Cell Mass (ICM) as a group of cells that intermingles with the closely related epiblast precursor. All ICM cells contain Oct4, a key transcription factor that is first expressed at the morula stage. In vitro, the epiblast precursor is most closely represented by the well-characterized embryonic stem (ES) cell lines that maintain the expression of Oct4, but analogous ExEn precursor cell lines are not known and it is unclear if they would express Oct4. METHODOLOGY/PRINCIPAL FINDINGS:Here we report the isolation and characterization of permanently proliferating Oct4-expressing rat cell lines ("XEN-P cell lines"), which closely resemble the ExEn precursor. We isolated the XEN-P cell lines from blastocysts and characterized them by plating and gene expression assays as well as by injection into embryos. Like ES cells, the XEN-P cells express Oct4 and SSEA1 at high levels and their growth is stimulated by leukemia inhibitory factor, but instead of the epiblast determinant Nanog, they express the ExEn determinants Gata6 and Gata4. Further, they lack markers characteristic of the more differentiated primitive/visceral and parietal ExEn stages, but exclusively differentiate into these stages in vitro and contribute to them in vivo. CONCLUSIONS/SIGNIFICANCE:Our findings (i) suggest strongly that the ExEn precursor is a self-renewable entity, (ii) indicate that active Oct4 gene expression (transcription plus translation) is part of its molecular identity, and (iii) provide an in vitro model of early ExEn differentiation

Evaluation of canine and ovine oviducts for maturation of canine oocytes from antral follicles

Due to the character of the original source materials and the nature of batch digitization, quality control issues may be present in this document. Please report any quality issues you encounter to [email protected], referencing the URI of the item.Includes bibliographical references (leaves 50-57).Issued also on microfiche from Lange Micrographics.The goal of this study was to determine whether canine or ovine oviducts improve the maturation of canine oocytes obtained from antral follicles over an in vitro system. The objective of the first experiment was to evaluate the canine oviduct for its ability to promote maturation of canine oocytes obtained from the ovaries following spay surgery of the donor bitches. The objective of the second experiment was to evaluate ovine oviducts for their ability to mature similarly obtained canine oocytes. All oocytes utilized in both the first and second experiment were obtained from bitches in proestrus or early estrus. Immediately after the ovaries were collected from the spayed bitches they were placed in warm physiological saline solution and transported (3h) to the laboratory for retrieval of the oocytes. After retrieval, the oocytes were maintained in a warm Hepes buffered medium until being surgically transferred into oviducts or placed into the in vitro control culture medium. Of the 156 oocytes recovered from canine oviducts and adjusted for the number of ovulated oocytes, one matured to metaphase II (MII), seven matured to metaphase I (MI), and two had undergone only germinal vessicle breakdown (GVBD). The 198 in vitro control oocytes for canine replicates contained six that matured to MII, 11 that matured to MI, and six that underwent only GVBD. Transfer of canine oocytes into the sheep oviduct yielded three metaphase II oocytes, two MI, and three that underwent only GVBD out of 138 recovered. The 181 in vitro control oocytes for the sheep replicates contained 14 that matured to metaphase II, nine to MI, and six that underwent only GVBD. In conclusion, both the canine and ovine oviducts support the maturation of canine oocytes, but the level of maturation was not an improvement over the in vitro control. The maturation rate of canine oocytes in ovine oviducts was lower (p<.05) than the maturation rate of in vitro control oocytes. Incubating the extirpated oocytes in the dog oviduct provided no significant difference (p<.05) in numbers of oocytes matured to MII compared to in vitro controls

List of primers used in this study.

<p>List of primers used in this study.</p

Properties of rat blastocyst outgrowths.

<p>(A) Phase contrast photographs showing stages of WKY rat blastocyst outgrowths kept on mitomycin-treated primary rat embryo fibroblasts (PREF). The outgrowths were initially smooth and compact (left), but converted to XEN morphology (right) ∼10 days after blastocyst plating if not passaged, or a few days later if mechanically disaggregated into smaller clumps. Regardless of when the conversion occurred, it was fast (<24 hours) and went through a stage of intermediate morphology (middle). (B) Loss and re-expression of Oct4 mRNA in WKY rat blastocyst outgrowths. In these experiments, the outgrowths were not passaged and showed compact, smooth morphology before day 10, but XEN morphology thereafter. At the indicated days, the outgrowths were individually harvested for RT-PCR analysis, using rat-specific primers for Oct4 and hypoxanthine phosphoribosyl transferase (Hprt) cDNAs. The Oct4 and Hprt cDNAs were amplified in the same reaction; none of the primers amplified intronless products from genomic DNA (not shown). No amplification was achieved when using mouse-specific primers (not shown). Day 0 = blastocyst; W, water control. (C) Semi-quantitative assessment of Oct4 mRNA level. Rat blastocysts (E4.5, strain WKY), XEN-P line RX1, primary XEN-like blastocyst outgrowths (strain WKY), rat embryo fibroblast line Li 1 (feeder for RX1), and PREF (feeder for primary rat cells) were analyzed for Oct4 and Hprt mRNAs by subjecting 10-fold serial dilutions of the RT reactions to PCR. (D) LIF effect (1,000 u/ml) on the formation of secondary XEN-like cell colonies from primary rat blastocyst outgrowths (WKY). Primary cells were seeded at ∼100–500 cells/well onto feeder line Li 1. 6 independent experiments. Similar results were obtained with rat strain BDIX.</p

Growth behavior and comparative embryonic lineage marker analysis of rat XEN-P cell lines.

<p>(A) Phase contrast photo showing characteristic morphology of rat XEN-P cell lines growing on rat embryo fibroblast feeder. Colonies obtained by low-density plating typically contained round, refractile cells at their fringes and epithelial cells inside (inset). (B) Representative photos illustrating that LIF (1000 u/ml) increased colony diameter and frequency (crystal violet staining) (line RX1). Similar results were obtained with line RX2 (strain BDIX). (C) RT-PCR analysis showing that rat XEN-P cell lines exhibit a mixed embryonic lineage marker profile. Rat XEN-P cell lines (RX1, RX2, RX5) were compared with mouse XEN cell lines (MX4, MX6), a mouse ES cell line (D3), a trophectoderm-like rat cell line (B10), a rat embryo fibroblast cell line (Li1) used as feeder for the XEN-P cell lines, and primary mouse embryo fibroblasts (MEF) used as feeders for mouse XEN and ES cells. Lines D3 and B10 have been described before <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007216#pone.0007216-Doetschman1" target="_blank">[43]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007216#pone.0007216-EppleFarmer1" target="_blank">[13]</a>. 2 µg of RNA per sample were reverse-transcribed or not (-RT), followed by PCR using dual-specific (rat = mouse) primers. For Gata6, Foxa2, and Dab2, two dilutions of the RT reaction were subjected to PCR for semi-quantitative comparison. (D) Western blot analysis of XEN-P (RX1), mouse XEN (MX4), and feeder (MEF, Li1) cell lines. 40 µg of cell protein were loaded per lane. (E) Northern blot analysis of XEN-P (RX1), mouse XEN (MX4), mouse ES (D3), and feeder (MEF, Li1) cell lines. 5 µg of total RNA were loaded per lane. (F) Western blot analysis for Oct4 in rat XEN-P (RX1), mouse XEN (MX4), mouse ES (D3), and feeder (MEF, Li1) cell lines, using a monoclonal anti-Oct4 antibody. 50 µg (top) or the indicated amounts (bottom) of cell protein were loaded. RX1 samples from two passages (P39, P40) were analyzed (bottom). Similar results were obtained with a polyclonal antibody (not shown). (G) Transient expression of mouse Oct4 gene-based LacZ reporter gene GOF9 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007216#pone.0007216-Yeom1" target="_blank">[53]</a> by rat XEN-P and mouse ES but not mouse XEN cell lines. Histochemical stainings of lines D3, MX4, and RX1 (similar results were obtained with line RX2). Non-transfected cells did not show LacZ staining (not shown). When comparing the frequencies of reporter gene expression in mouse ES vs. rat XEN-P cell lines, keep in mind that only a subpopulation in the rat cell lines highly expresses the endogenous <i>Oct4</i> gene (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007216#pone-0007216-g003" target="_blank">Fig. 3</a>).</p

Contributions of rat XEN-P cell lines to postimplantation embryos.

<p>Representative fluorescence (A–C) and bright field (A'–C') photographs demonstrating in vivo contributions of microinjected rat cells to (A, A') parietal yolk sac of an 11.5 dpc rat conceptus (inset showing magnification); (B, B') visceral endoderm of an 8.5 dpc rat conceptus; (C, C') visceral endoderm (arrowheads; one patch magnified in inset) of an ∼7 dpc mouse conceptus. Pregnancy timing is distorted by the embryo manipulations and therefore only approximate.</p

Quantitative RT-PCR for Oct4 in whole-culture (*) and micro- (**) XEN-P cell line samples.

<p>Samples (numbers in brackets) were RNA-extracted, RNA preparations were DNAse-treated and quantified in duplicate by real-time RT-PCR using dual-specific (mouse = rat) primers; controls without reverse transcriptase did not yield a product. Data (Means±SEM) were normalized to Hprt mRNA and expressed as fold of the level in ES cells, i.e. ES cell level is set as 1.</p>A<p>, ^B, two groups of microsamples with high and moderate Oct4 mRNA expression, respectively. Two experiments labeled ^C were corrected for feeder cell RNA; the other measurements are slight underestimates.</p