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.

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.

Trophoblast antigenicity demonstrated by altered challenge graft survival.

Adult C57BL/6J male mice received either a primary ectopic transplant or a primary and a challenge ectopic transplant of trophoblast tissue obtained from the ectoplacental cones of 7(1/2)-day-old C3H/HeJ x C3H/HeJ embryos. Gross and histologic examinations of these grafts at 5, 7, and 12 days of growth indicated that the challenge grafts were inhibited in growth; there were a smaller percentage of grossly successful grafts, smaller hemorrhagic reactions, fewer viable cells at all stages of growth, and better host containment of the colony. The evidence indicates that exposure to pure trophoblast alters an animal's subsequent reaction to grafts syngeneic with the original trophoblast and can best be explained as evidence of immunologic sensitization of the host.

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.

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.

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.

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.

Pregnancy-induced hyporesponsiveness to paternal alloantigens. II. Factors affecting altered expressions of immunity in parous rats.

It was previously demonstrated that female Fischer rats, primiparous by allogeneic DA males, produced only low levels of agglutinating antibody to a challenge skin graft bearing paternal alloantigens, although graft rejection was unimpaired. In this report, the phenomenon of pregnancy-induced hyporesponsiveness was further investigated with regard to factors responsible for its induction and maintenance. Increasing parity by DA males resulted in increased suppression of the Fischer females' agglutinating antibody production postpartum to a challenge skin graft bearing paternal antigens. Moreover, multiparity also resulted in impaired graft rejection, with greater numbers of litters causing more prolonged survival. Either humoral or cellular hyporesponsiveness, or both, persisted for several weeks or even months in many females. Removal of the iliac lymph nodes prior to pregnancy resulted in less severe suppression which was not maintained through the observation period (21 days postgrafting), whereas splenectomy either before mating or on day 7 of pregnancy had no effect. Tubal-ligated females exposed to multiple DA ejaculates, without pregnancy, manifested weakened humoral immunity similar to that of primiparous females, whereas those exposed to (Fischer X DA)F1 hybrid trophoblast multiple times in ectopic sites had not only somewhat suppressed antibody responses but also significantly impaired capacity to reject the challenge allograft. The potential roles of various antigenic stimuli and the complexities of intrauterine antigen processing during gestation are discussed in relation to this naturally induced allosuppression phenomenon.

Trophoblast growth in the lungs of mice.

Trophoblast giant cells grow from ectoplacental cones of 7 1/2 days' gestation that are implanted in the lung from the surface, intrabronchially, or via the circulation. When blastocysts of 3 1/2 days' gestation are implanted in lung, their behavior varies according to the route of implantation and the ages of the recipient mice. Blastocysts produced trophoblast giant cells when implanted in the bronchial tree. Trophoblast giant cells were not seen in adult lungs when blastocysts were introduced via the circulation. However, they were seen in 12-day-old mice injected in the same way 10 days previously. Something is apparently liberated from adult lung vessels which interacts with the blastocyst or zona pellucida and causes rapid degeneration of trophoblast. Experiments designed to extract this agent are in progress in the hope that the agent may be useful in the treatment of human trophoblastic disease.

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.

[Different modes of cell reproduction in the placental differentiation of mammals]. / Razlichnye puti reproduktsii kletok pri differentsirovke platsenty mlekopitaiushchikh.

Summarized is the total evidence on the role of various modes of cell reproduction in the course of differentiation of various trophoblast population cells. The giant trophoblast cells, reaching a tremendously high (for mammals) degree of ploidy (512-4096c), blast cells, reaching a tremendously high for (mammals) degree of ploidy (512-4096c), give an example of cells that display a "non-classical" form of polyteny with not regular polyteny in chromosomes. For the trophoblast cells, a period of easy recognition with not regular, polyteny in chromosomes. For display a "non-classical form of polyteny with not regular polyteny in chromosomes. For the trophoblast cells, a period of easy recognition polytene chromosome has been established timed to definite stages of cell differentiation and of the polytene nucleus cycle. The start of phagocytic activity in trophoblast cells coincides in time with their transition to endoreduplication, both the processes terminating also simultaneously. It does not seem unlikely that such that each a correlation may appear due to a strict programmed appearance of numerous features of trophoblast cells, including their lifespan both in situ and under condition of -transplantation to different organs. Biological importance of various ways of reproduction in trophoblast cells (mitosis, non-completed polyploidizing mitosis, endoreduplication) in various parts of placenta is discussed in relation to their roles in development and functioning of this organo.

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.

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.