ERp29 affects the migratory and invasive ability of human extravillous trophoblast HTR-8/SVneo cells via modulating the epithelial-mesenchymal transition.

Dysfunction of trophoblast metastasis into the endometrium is the main cause of pre-eclampsia (PE); however, the factors affecting this process are still unclear. In this study, we found that endoplasmic reticulum protein 29 (ERp29), one molecular chaperone of the endoplasmic reticulum, was aberrantly upregulated in the placenta of pre-eclamptic patients compared with healthy controls. Then, an in vitro study using human extravillous trophoblast HTR-8/SVneo cells showed that ERp29 upregulation could inhibit the migratory and invasive ability of HTR-8/SVneo cells, while ERp29 downregulation had the opposite effect. Mechanical experiments confirmed that ERp29 blocked trophoblast metastasis via inhibiting the process of epithelial-mesenchymal transition and affecting the Wnt/ß-catenin signaling pathway. In conclusion, this study revealed the important role of ERp29 in trophoblast metastasis and improved the mechanical understanding of PE occurrence.

Transplantation of human villous trophoblasts preserves cardiac function in mice with acute myocardial infarction.

Over the past decade, cell therapies have provided promising strategies for the treatment of ischaemic cardiomyopathy. Particularly, the beneficial effects of stem cells, including bone marrow stem cells (BMSCs), endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs), have been demonstrated by substantial preclinical and clinical studies. Nevertheless stem cell therapy is not always safe and effective. Hence, there is an urgent need for alternative sources of cells to promote cardiac regeneration. Human villous trophoblasts (HVTs) play key roles in embryonic implantation and placentation. In this study, we show that HVTs can promote tube formation of human umbilical vein endothelial cells (HUVECs) on Matrigel and enhance the resistance of neonatal rat cardiomyocytes (NRCMs) to oxidative stress in vitro. Delivery of HVTs to ischaemic area of heart preserved cardiac function and reduced fibrosis in a mouse model of acute myocardial infarction (AMI). Histological analysis revealed that transplantation of HVTs promoted angiogenesis in AMI mouse hearts. In addition, our data indicate that HVTs exert their therapeutic benefit through paracrine mechanisms. Meanwhile, injection of HVTs to mouse hearts did not elicit severe immune response. Taken together, our study demonstrates HVT may be used as a source for cell therapy or a tool to study cell-derived soluble factors for AMI treatment.

Cardiac repair in a mouse model of acute myocardial infarction with trophoblast stem cells.

Various stem cells have been explored for the purpose of cardiac repair. However, any individual stem cell population has not been considered as the ideal source. Recently, trophoblast stem cells (TSCs), a newly described stem cell type, have demonstrated extensive plasticity. The present study evaluated the therapeutic effect of TSCs transplantation for heart regeneration in a mouse model of myocardial infarction (MI) and made a direct comparison with the most commonly used mesenchymal stem cells (MSCs). Transplantation of TSCs and MSCs led to a remarkably improved cardiac function in contrast with the PBS control, but only the TSCs exhibited the potential of differentiation into cardiomyocytes in vivo. In addition, a significantly high proliferation level of both transplanted stem cells and resident cardiomyocytes was observed in the TSCs group. These findings primary revealed the therapeutic potential of TSCs in transplantation therapy for MI.

Ectopic Trophoblast Allografts in the Horse Resist Destruction by Secondary Immune Responses.

Invasive trophoblast from Day 34 horse conceptuses survives in extrauterine sites in allogeneic recipients that are immunologically naive to donor major histocompatibility complex class I antigens. The ectopic trophoblast retains its in utero characteristics, including similar lifespan, physiologic effect of its secreted product (equine chorionic gonadotropin) upon the recipient's ovaries, and induction of host immune responses. Immunologic memory has not been considered previously in this experimental system. We hypothesized that primary exposure to ectopic trophoblast would affect the recipient's immune status such that the survival time of subsequent transplants would be altered. Secondary transplant lifespans could be shortened by destructive memory responses, as has been observed in ectopic trophoblast studies in rodents, or lengthened, as occurs when male skin grafts follow multiple syngeneic pregnancies in mice. Eight mares received two closely spaced trophoblast transplants. Both grafts for each recipient were obtained from conceptuses sired by the same stallion to provide consistency in histocompatibility antigen exposure. Donor stallions were major histocompatibility complex class I homozygotes. Cytotoxic antibody production was tracked to monitor recipients' immune responses to the transplants. Detection of serum equine chorionic gonadotropin was used as a proxy for transplant lifespan. There was no significant difference between the distributions of primary and secondary transplant lifespans, despite evidence of immunologic memory. These data demonstrate that secondary ectopic trophoblast transplants in horses do not experience earlier destruction or prolonged survival following immune priming of recipients. Mechanisms responsible for the eventual demise of the transplants remain unperturbed by secondary immune responses or chronic antigenic exposure.

Progesterone-induced blocking factor differentially regulates trophoblast and tumor invasion by altering matrix metalloproteinase activity.

Invasiveness is a common feature of trophoblast and tumors; however, while tumor invasion is uncontrolled, trophoblast invasion is strictly regulated. Both trophoblast and tumor cells express high levels of the immunomodulatory progesterone-induced blocking factor (PIBF), therefore, we aimed to test the possibility that PIBF might be involved in invasion. To this aim, we used PIBF-silenced or PIBF-treated trophoblast (HTR8/Svneo, and primary trophoblast) and tumor (HT-1080, A549, HCT116, PC3) cell lines. Silencing of PIBF increased invasiveness as well as MMP-2,-9 secretion of HTR8/SVneo, and decreased those of HT-1080 cells. PIBF induced immediate STAT6 activation in both cell lines. Silencing of IL-4Rα abrogated all the above effects of PIBF, suggesting that invasion-related signaling by PIBF is initiated through the IL-4Rα/PIBF-receptor complex. In HTR-8/SVneo, PIBF induced fast, but transient Akt and ERK phosphorylation, whereas in tumor cells, PIBF triggered sustained Akt, ERK, and late STAT3 activation. The late signaling events might be due to indirect action of PIBF. PIBF induced the expression of EGF and HB-EGF in HT-1080 cells. The STAT3-activating effect of PIBF was reduced in HB-EGF-deficient HT-1080 cells, suggesting that PIBF-induced HB-EGF contributes to late STAT3 activation. PIBF binds to the promoters of IL-6, EGF, and HB-EGF; however, the protein profile of the protein/DNA complex is different in the two cell lines. We conclude that in tumor cells, PIBF induces proteins, which activate invasion signaling, while-based on our previous data-PIBF might control trophoblast invasion by suppressing proinvasive genes.

Functions of ectopically transplanted invasive horse trophoblast.

The invasive and fully antigenic trophoblast of the chorionic girdle portion of the equine fetal membranes has the capacity to survive and differentiate after transplantation to ectopic sites. The objectives of this study were to determine i) the survival time of ectopically transplanted allogeneic trophoblast cells in non-pregnant recipient mares, ii) whether equine chorionic gonadotropin (eCG) can be delivered systemically by transplanted chorionic girdle cells, and iii) whether eCG delivered by the transplanted cells is biologically active and can suppress behavioral signs associated with estrus. Ectopically transplanted chorionic girdle survived for up to 105 days with a mean lifespan of 75 days (95% confidence interval 55-94) and secreted sufficient eCG for the hormone to be measurable in the recipients' circulation. Immunohistochemical labeling of serial biopsies of the transplant sites and measurement of eCG profiles demonstrated that graft survival was similar to the lifespan of equine endometrial cups in normal horse pregnancy. The eCG secreted by the transplanted cells induced corpora lutea formation and sustained systemic progesterone levels in the recipient mares, effects that are also observed during pregnancy. This in turn caused suppression of estrus behavior in the recipients for up to 3 months. Thus, ectopically transplanted equine trophoblast provides an unusual example of sustained viability and function of an immunogenic transplant in a recipient with an intact immune system. This model highlights the importance of innate immunoregulatory capabilities of invasive trophoblast cells and describes a new method to deliver sustained circulating concentrations of eCG in non-pregnant mares.

Identification of multipotent cytotrophoblast cells from human first trimester chorionic villi.

In this article we used immunohistochemistry and FACS analyses to show that cells expressing markers typical of human stem cells such as SSEA4, OCT-4, ALP, and CD117 are present within the cytotrophoblastic tissue of human fetal chorionic villus samples (CVSs). After immunoselection of CV cells for SSEA4, FACS analyses showed an increased number of cells positive for OCT-4 and ALP and a small percentage (around 4%) of side population (SP) cells. In the same cell population, RT-PCR indicated the presence of OCT-4, NANOG, and SOX2 transcripts, also typical of stem cells. Depending on the in vitro conditions, a subset of SSEA4+ cells formed colonies resembling hESCs, with limited self renewal ability. At the same time, these cells were able to differentiate in vitro into derivatives of all three germ layers. When inoculated into immunocompromised mice, SSEA4+ cells did not form teratomas but were able to populate depleted hematopoietic tissues. Moreover, after injection into mouse blastocysts, they were incorporated into the inner cell mass and could be traced into several tissues of the adult chimeric mice. Finally, we show that SSEA4+ cells isolated from fetuses affected by Spinal Muscular Atrophy (SMA) can be genetically corrected with high efficiency in culture by Small Fragment Homologous Recombination (SFHR), a gene targeting approach. Taken together, our results indicate that SSEA4+ cells obtained from human CVSs contain a subpopulation of multipotent cells that we propose to name Human Cytotrophoblastic-derived Multipotent Cells (hCTMCs). These cells may be a safe and convenient source of cells for cell-based therapy, as well as an ideal target for in utero fetal gene therapy.

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.

Modeling trophoblast differentiation using equine chorionic girdle vesicles.

The chorionic girdle of the equine conceptus is comprised of specialized trophoblast cells which, at day 36-38 of equine pregnancy, gain an invasive phenotype and invade the endometrium to form endometrial cups. Studies of equine endometrial cups remain difficult to perform because of the invasive techniques required to obtain cup tissue and because sampling requires termination of the pregnancy. In this study we developed a system to model trophoblast differentiation and trophoblast-immune interactions in vitro and in vivo. We utilized a method of culturing chorionic girdle pieces in serum-free medium to promote spontaneous formation of vesicle structures enriched for terminally differentiated binucleate cells that secreted equine chorionic gonadotrophin (eCG). Immunohistochemical staining and scanning electron microscopy showed that the cells of the vesicles closely resembled the outer layers of chorionic girdle immediately prior to invasion. Chorionic girdle vesicles were harvested after 72h in culture and ectopically transplanted via injection into the vulvar mucosa of recipient mares. At 7, 14, 21 and 28days after transplantation, biopsies of the injection sites were obtained. Immunohistochemical labeling of cryostat sections of the biopsies with a panel of monoclonal antibodies to horse trophoblast molecules demonstrated survival, differentiation, and presence of trophoblast cells for at least 21days. Serial sections of the biopsies labeled with antibodies to the equine lymphocyte surface markers CD4 and CD8, together with lymphocyte microcytotoxicity assays, revealed that the recipients mounted both cellular and humoral antibody immune responses to the transplanted trophoblast cells. This new method for culturing equine chorionic girdle trophoblast cells, and for transplanting trophoblast vesicles to ectopic sites, should allow identification of key aspects of trophoblast differentiation and the interactions that occur between invasive trophoblast and the maternal immune system.

Trophoblast stem cells rescue placental defect in SOCS3-deficient mice.

Stem cells have important clinical and experimental potentials. Trophoblast stem (TS) cells possess the ability to differentiate into trophoblast subtypes in vitro and contribute to the trophoblast lineage in vivo. Suppressor of cytokine signaling 3 (SOCS3) is a negative regulator of cytokine signaling. Targeted disruption of SOCS3 revealed embryonic lethality on E12.5; it was caused by placental defect with enhanced leukemia inhibitory factor receptor signaling. A complementation of the wild-type (WT) placenta by using tetraploid rescue technique showed that the embryonic lethality in SOCS3-deficient embryo was due to the placental defect. Here we demonstrate that TS cells supplementation rescues placental defect in SOCS3-deficient embryos. In the rescued placenta, TS cells were integrated into the placental structure, and a substantial structural improvement was observed in the labyrinthine layer that was disrupted in the SOCS3-deficient placenta. Importantly, by supplying TS cells, living SOCS3-deficient embryos were detected at term. These results indicate a functional contribution of TS cells in the placenta and their potential application.

Connexins and trophoblast cell lineage development.

The mouse is a valuable model for studying basic mechanisms of gene regulation in trophoblast cell lineage differentiation. Elements of placental development are conserved across species, including trophoblast proliferation, differentiation, migration, and vessel invasion. Among the regulatory processes, direct intercellular communication between trophoblast cells via gap junction channels seems to play a crucial role in placental development and physiology. Here we describe in detail the generation of trophoblast stem (TS) cell lines from connexin-deficient mice. The design of differentiation and proliferation assays are specified including marker gene sets which are important for analyzing and comparing the differentiation capacity of the connexin-deficient TS cell lines. Furthermore, we show that TS cells are capable of forming tumors after subcutaneous injection into nude mice, providing the opportunity to investigate trophoblast invasion into host vessels in vivo.

Production of capsular material by equine trophoblast transplanted into immunodeficient mice.

A novel xenogeneic transplantation approach was used to determine whether it is embryonic or maternal tissue that produces the material that gives rise to the mucin-like glycoprotein of the equine embryonic capsule. Endometrial biopsy samples and conceptuses from six mares at days 13-15 after ovulation were prepared as 1 mm(3) grafts of endometrium, trophoblast and capsule for transplantation, alone or in combination, into various sites in 88 immunodeficient (severe combined immunodeficient or RAG2/gamma(c) double mutant) mice. The overall recovery rate of grafts was over 50%, reaching 100% with experience and use of the renal subcapsular space exclusively. Periodic acid-Schiff (PAS) staining demonstrated capsule-like extracellular glycoprotein secretions at the graft site in 11 of 22 sites examined. Strong PAS-positive reactions (5-7 microm thick) were found in four of six sites containing trophoblast alone, five of six endometrium plus trophoblast sites, and zero of eight grafts of endometrium alone. Two recovered grafts of capsule were also PAS-positive. The secreted glycoprotein was identified as equine embryonic capsule material by using a monoclonal antibody (mAb) specific to equine capsule (mAb OC-1) in two experiments. In the first, in cryosections, this antibody bound to 19 of 19 recovered trophoblast graft secretions (including those in 12 from mice that had not received endometrium at any site), ten of ten recovered endometrium plus trophoblast grafts, and zero of 12 recovered endometrial grafts from mice in which trophoblast had been grafted to the same site or another site in the same mouse. In the second experiment, in paraformaldehyde-fixed sections of grafts from 11 mice, specific staining, identical to that shown by grafted capsule, was obtained with grafts of trophoblast (both alone and in combination with endometrium) but not with grafts of endometrium. These results support the contention that trophoblast is the principal source of equine embryonic capsule. In addition, they demonstrate that xenogeneic grafting is a useful means of culturing endometrium and conceptus tissues outside the mare when in vitro techniques do not suffice.

Normalization of blood glucose after islet cografting with placental tissues in diabetic mice.

A fetus in the uterus is not rejected at any time during the entire gestational period, even without the administration of immunosuppressive agents, though fetus is a kind of allograft. This prevention of rejection is considered to be associated with the presence of placental tissues. This hypothesis was tested by the allografting of islets together with placental tissues (trophoblasts) in streptozotocin (STZ)-induced diabetic mice. Placentae were harvested from the mice at the 14th postgestation day by being peeled off carefully and with the maternal decidua left behind, and cut into small pieces. Five hundred freshly isolated islets together with placental tissues obtained from ICR mice were placed under the left kidney capsule of STZ-induced diabetic C57BL/6J mice. The nonfasting blood glucose level was reduced from 477 +/- 41 mg/dl at the time of pretransplant to that of the intact normal mice (161 +/- 18 mg/dl) soon after the cografting, and did not return to the pretransplant level before the 14th posttransplant day. The grafting of the same number of islets alone and/or liver tissues dropped the blood glucose level, but not to that of the intact normal mice. It returned to the pretransplant level within 1 week. This is the first successful prolongation of survival of allografted islets without immunosuppressive agents through cotransplantation of allogenic placental tissues. The underlying mechanism remains to be clarified.

Trophoblastic peritoneal implants after laparoscopic treatment of ectopic pregnancy.

Persistent trophoblastic activity after salpingostomy for ectopic pregnancy implies the presence of intra-abdominal trophoblastic tissue, usually within the fallopian tube. We report a case of disseminated trophoblastic peritoneal implants, presenting as hemoperitoneum three weeks after laparoscopic salpingectomy. Only 23 such cases have been reported. Surgical treatment of ectopic pregnancy, especially by the laparoscopic technique, may cause intraperitoneal spread and reimplantation of trophoblastic tissue. Precautions for minimizing this complication are discussed.

Ectopic transplantation of equine invasive trophoblast.

A system for transplanting invasive equine trophoblast (i.e., chorionic girdle) to ectopic sites has been developed as a means to study the differentiation of this tissue and to assess maternal immune responses to the conceptus tissue in a site outside the uterus. Chorionic girdle was isolated from Day 33 to 34 conceptuses and surgically placed into the vulvar mucosa or subdermal skin of recipient mares. Biopsy specimens of the graft sites for immunohistochemical staining were taken at weekly or biweekly intervals after grafting. Serum samples were collected from each recipient and tested for antibody to donor major histocompatibility complex (MHC) class I antigens using the lymphocyte microcytotoxicity assay. Transplanted trophoblast cells expressed differentiation markers associated with invading chorionic girdle and endometrial cup cells. The transplanted trophoblast cells were also labeled by an antibody to eCG. Strong cellular and humoral immune responses to the transplanted tissue were mounted by the recipients, similar to those occurring during normal equine pregnancy. Despite these responses, the invasive trophoblast transplants survived for at least 28 days after grafting and downregulated MHC class I antigens, as do the mature endometrial cup cells in equine pregnancy. These findings suggest that invasive equine trophoblast has the capacity to differentiate fully in equine nonuterine tissues, and that it can evade maternal immune responses independent of the physiological state of pregnancy and in sites other than the uterus.

Neural transplantation of the rat midgestation trophoblast.

The trophoblast may serve as an example of tissue that is endowed with invasive properties under physiological conditions. Invasiveness of the trophoblast is enabled by the secretion of various proteases capable of degrading extracellular matrix components. Trophoblast invasive behavior is strictly controlled by anti-invasive factors produced by uterine decidual cells. To assess invasive abilities of trophoblast cells we have transplanted E9 and E14 rat trophoblast (i.e. the trophoblast obtained on embryonic day 9 and 14) into the brain of adult rats. The brain parenchyma as an immunologically privileged site is suitable for acceptance of grafts of different tissues. Moreover, the trophoblast placed into the CNS lacks inhibitory influence of anti-invasive factors that normally regulate trophoblast invasivity in the course of intrauterine gestation. To visualize migration of grafted cells the nuclei of the trophoblast were labelled with bromodeoxyuridine prior to neural grafting. The transplant of E9 and E14 trophoblast cells obtained a blood nourishment from host vessels. A proper vascularization is necessary for a further transplant growth. The transplant contained labyrinthine trophoblast cells and giant cells that are typical for the rat placenta. Vital trophoblast cells were found in all grafts whose age did not exceed a lifespan of normal rat trophoblast cells i.e. 21-22 days. In the centre of the graft, no blood vessels were observed. Interstitial spaces of neighbouring trophoblast cells were filled with the host blood and morphology of these spaces mimicked lacunae of the placental trophoblast. E9 and E14 rat trophoblast continued to differentiate after transplantation into the CNS of adult rats. Histological structure of the grafts were compared with microscopical morphology of the normal rat placenta. E9 and E14 trophoblast is considerably differentiated and it does not invade a neighbouring tissue. Trophoblast cells located at t the graft periphery may migrate on free surfaces but they do not invade through the host parenchyma. Migration occurs at a limited distance from the transplant and the cells remain in a close contact with other trophoblast cells in the graft via their cytoplasmatic processes. The ability to lyse host blood vessels and form vascular lacunae is well preserved in E9 and E14 trophoblast after grafting into the CNS. This ability is necessary for a proper transport of nutrients from the host blood stream to fetal tissue that normally occurs in the placenta.

Maintenance of the corpus luteum after uterine transfer of trophoblastic vesicles to cyclic cows and ewes.

One or two trophoblastic vesicles (0.4-2 mm diam.) from cow (Day 14) or ewe (Day 11-13) embryos without their disc were transferred, after culture for 24 h, into recipients. Each vesicle was transferred into the uterine horn ipsilateral to the CL by the cervical route in heifers and surgically in ewes on Day 12 of the oestrous cycle. In cows, daily measurements of plasma progesterone concentrations and checks for return to oestrus showed that the CL was maintained in 8 out of 12 recipients. These 8 cows had 25- to 37-day cycles while 4 recipient heifers returned to oestrus normally. Three recipients with an extended cycle were slaughtered. The dissected uterus showed that trophoblastic vesicles had developed in the uterine horns. In ewes, the serum progesterone curve, determined in each recipient, showed that the CL was maintained in 7 out of 12 recipients. These 7 ewes had 20- to 54-day cycles and the other 5 ewes had a normal cycle of 15-19 days comparable to that of 17.0 +/- 0.5 days for the 6 control ewes. Whenever the CL was maintained, high blood progesterone levels were followed by rapid luteolysis. In cattle and sheep, therefore, a trophoblastic vesicle transferred into the uterus can develop in vivo, secreting the embryonic signals when there is no embryonic disc control and transforming the cyclic CL into a CL of pregnancy in about 60% of the cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Recruitment of cytotoxic cells by ectopic grafts of xenogeneic, but not allogeneic, trophoblast.

In previous studies we showed that survival of Mus caroli embryos in the Mus musculus uterus was dependent on trophoblast genotype; M musculus trophoblast was protective while M caroli trophoblast was not. In this report we compare the immune response of M musculus to ectopic grafts of pure trophoblast of M caroli and M musculus genotypes to elucidate further differences between the properties of trophoblast from the two species that might account for the failure of M caroli trophoblast to protect the fetus. Grafts of M caroli or DBA/2J ectoplacental cone (EPC) or embryonic tissue (E) were placed beneath the renal capsule of Ha(ICR) mice that were untreated or had been immunized with M caroli lymphocytes or with P815 mastocytoma cells. No consistent morphological difference could be demonstrated between the xenografts and allografts by gross or histological examination. Cellular infiltration of allogeneic and xenogeneic EPC graft sites occurred in immunized mice within 5 days of grafting. Cells specifically cytotoxic to M caroli lymphoblasts were isolated from the xenogeneic graft sites, but no allogeneically sensitized cytotoxic cells could be demonstrated. These results show that xenoantigens expressed on trophoblast induce a cytotoxic immune response more readily than alloantigens.