Development of properly-polarized trophoblast stem cell-derived organoids to model early human pregnancy.

The development of human trophoblast stem cells (hTSC) and stem cell-derived trophoblast organoids has enabled investigation of placental physiology and disease and early maternal-fetal interactions during a stage of human pregnancy that previously had been severely restricted. A key shortcoming in existing trophoblast organoid methodologies is the non-physiologic position of the syncytiotrophoblast (STB) within the inner portion of the organoid, which neither recapitulates placental villous morphology in vivo nor allows for facile modeling of STB exposure to the endometrium or the contents of the intervillous space. Here we have successfully established properly-polarized human trophoblast stem cell (hTSC)-sourced organoids with STB forming on the surface of the organoid. These organoids can also be induced to give rise to the extravillous trophoblast (EVT) lineage with HLA-G + migratory cells that invade into an extracellular matrix-based hydrogel. Compared to previous hTSC organoid methods, organoids created by this method more closely mimic the architecture of the developing human placenta and provide a novel platform to study normal and abnormal human placental development and to model exposures to pharmaceuticals, pathogens and environmental insults. Motivation: Human placental organoids have been generated to mimic physiological cell-cell interactions. However, those published models derived from human trophoblast stem cells (hTSCs) or placental villi display a non-physiologic "inside-out" morphology. In vivo , the placental villi have an outer layer of syncytialized cells that are in direct contact with maternal blood, acting as a conduit for gas and nutrient exchange, and an inner layer of progenitor, single cytotrophoblast cells that fuse to create the syncytiotrophoblast layer. Existing "inside-out" models put the cytotrophoblast cells in contact with culture media and substrate, making physiologic interactions between syncytiotrophoblast and other cells/tissues and normal and pathogenic exposures coming from maternal blood difficult to model. The goal of this study was to develop an hTSC-derived 3-D human trophoblast organoid model that positions the syncytiotrophoblast layer on the outside of the multicellular organoid.

Modeling human peri-implantation placental development and function†.

It is very difficult to gain a better understanding of the events in human pregnancy that occur during and just after implantation because such pregnancies are not yet clinically detectable. Animal models of human placentation are inadequate. In vitro models that utilize immortalized cell lines and cells derived from trophoblast cancers have multiple limitations. Primary cell and tissue cultures often have limited lifespans and cannot be obtained from the peri-implantation period. We present here two contemporary models of human peri-implantation placental development: extended blastocyst culture and stem-cell derived trophoblast culture. We discuss current research efforts that employ these models and how such models might be used in the future to study the "black box" stage of human pregnancy.

Comparison of extravillous trophoblast cells derived from human embryonic stem cells and from first trimester human placentas.

INTRODUCTION: Preeclampsia and other placental pathologies are characterized by a lack of spiral artery remodeling associated with insufficient invasion by extravillous trophoblast cells (EVT). Because trophoblast invasion occurs in early pregnancy when access to human placental tissue is limited, there is a need for model systems for the study of trophoblast differentiation and invasion. Human embryonic stem cells (hESC) treated with BMP4- differentiate to trophoblast, and express HLA-G, a marker of EVT. The goals of the present study were to further characterize the HLA-G(+) cells derived from BMP4-treated hESC, and determine their suitability as a model. METHODS: HESC were treated with BMP4 under 4% or 20% oxygen and tested in Matrigel invasion chambers. Both BMP4-treated hESC and primary human placental cells were separated into HLA-G(+) and HLA-G(-)/TACSTD2(+) populations with immunomagnetic beads and expression profiles analyzed by microarray. RESULTS: There was a 10-fold increase in invasion when hESC were BMP4-treated. There was also an independent, stimulatory effect of oxygen on this process. Invasive cells expressed trophoblast marker KRT7, and the majority were also HLA-G(+). Gene expression profiles revealed that HLA-G(+), BMP4-treated hESC were similar to, but distinct from, HLA-G(+) cells isolated from first trimester placentas. Whereas HLA-G(+) and HLA-G(-) cells from first trimester placentas had highly divergent gene expression profiles, HLA-G(+) and HLA-G(-) cells from BMP4-treated hESC had somewhat similar profiles, and both expressed genes characteristic of early trophoblast development. CONCLUSIONS: We conclude that hESC treated with BMP4 provide a model for studying transition to the EVT lineage.

Effect of leptin on mouse trophoblast giant cells.

Leptin plays a role in both energy homeostasis and reproduction, and it is required in early pregnancy. It stimulates metalloproteinase activity in cultured human trophoblasts and invasiveness of cultured mouse trophoblasts. Our goal has been to examine mechanisms that underpin the ability of leptin to promote trophoblast invasiveness in primary cultures of mouse trophoblasts. Leptin stimulated the phosphorylation of MEK (MAP2K1) but not signal transducer and activator of transcription 3 (STAT3) in the cultures, increased the concentration of the suppressor of cytokine signaling 3 (SOCS3) protein, and upregulated metalloproteinase activity. Microarray analysis revealed that leptin stimulated select genes with roles in cell motility, including Stmn, a gene linked to invasiveness in other cell types. There was also an increase in activity of several genes associated with MAPK and RhoGTPase signaling. In addition, leptin muted expression of genes correlated with terminal differentiation of trophoblast giant cells, including ones associated with the TGFbeta signaling pathway and endoreduplication of DNA, and upregulated selected prolactin-related family members. Feulgen staining of leptin-treated cells revealed a loss of cells with low ploidy. The data suggest that leptin accelerates disappearance of non-giant cells while inhibiting terminal differentiation of committed giant cells, possibly by maintaining cells in an intermediate stage of differentiation.

Human embryonic stem cells as models for trophoblast differentiation.

Trophectoderm is specified from pluripotent blastomeres at some time prior to blastocyst formation. Proliferating cytotrophoblast derived from trophectoderm is the forerunner of the entire trophoblast component of the mature human placenta, including extravillous cytotrophoblast and syncytiotrophoblast. Recently human embryonic stem cells (hESC) have been employed to study these events in an in vitro situation. Here we review some of the work in this emerging area of trophoblast biology. We concentrate primarily on a model in which colonies of hESC are exposed to BMP4 in stem cell growth medium lacking FGF2. Under both low (4%) and high (20%) O(2) conditions, differentiation proceeds unidirectionally towards trophoblast from the outside of the colonies inwards, with the progression fastest under high O(2). Immunohistochemical observations performed on whole colonies combined with microarray analysis of mRNA can be employed to track developmental transitions as they occur over time and in two-dimensional space as the cells respond to BMP4.

Leptin receptor expression increases in placenta, but not hypothalamus, during gestation in Mus musculus and Myotis lucifugus.

In addition to effects on appetite and metabolism, the hormone leptin is required for reproduction in mammals. Maternal plasma leptin is increased above non-pregnant levels in all mammals thus far examined, including humans. The increase in plasma leptin appears to result in part from upregulation of adipose leptin secretion (e.g., in mice), or from production and secretion of leptin from the placenta (e.g., in humans and some bats). The placenta may also modulate maternal leptin levels via production of a plasma leptin-binding protein (mice, humans). Thus, the placenta plays a coordinating role in regulation of maternal leptin during pregnancy. In this study, the hypothesis that the placenta is also a target organ for leptin in diverse taxa was tested by examining the expression of leptin receptors (Ob-R) in placentae from species of distantly related mammalian taxa, Mus musculus (the laboratory mouse) and Myotis lucifugus (the little brown myotis, also called the little brown bat). A partial sequence of M. lucifugus Ob-R cDNA was first obtained and found to share approximately 78-88% homology at the nucleotide level with known mammalian Ob-R cDNAs. Using probes and primers designed from this sequence, receptor expression was detected in numerous tissues of M. lucifugus, including placenta, which expressed two major receptor isoforms as judged by molecular size. In both species, Ob-R mRNA expression in placenta significantly increased from early to late gestation. Expression of Ob-R mRNA was not affected by cAMP treatment in vitro. The increase in Ob-R mRNA expression in placenta was specific, since Ob-R mRNA expression did not change during gestation in either species in hypothalamus, the major site of the central actions of leptin. Thus, Ob-R is expressed in placenta throughout gestation in mice and bats, and its expression increases over the course of gestation, which raises the possibility that leptin may exert temporally distinct effects on placental growth or function throughout gestation. Because similar placenta-specific changes in leptin receptor expression occurred in species from distantly related mammalian taxa which collectively comprise approximately 70% of all known mammalian species, it is possible that placental actions of leptin are conserved across mammals, even in those species (such as the Swiss-Webster strain of mouse) in which the placenta does not itself produce leptin.

[Erysipelas as a disease factor]. / Rotlauf als Faktorenkrankheit.

The infection with Erysipelas rhusiopathiae demonstrates that it is possible to characterize the significance of predisposing factors. The virulence of the agent is most important among the exogenous factors; it determines decisively the course of the disease: 1. paracute course as septicemia; 2. chronic course as polyarthritis; 3. subclinical course nearly without any symptoms. The immune status and the genotype of the host are predominant out of endogenous factors. The importance of immunity is known since a long time. A series of field observations supported the potential genetical influence in the pig. Within the hereditability an association to the MHC (in special genetic lines of rats to the RT 1 system of the MHC) was most recently determined in inbred laboratory animals. Additionally several environmental conditions, which can be summarized as stress, and as endogenous factor the age of the animals are relevant for the pathogenesis of the erysipelas infection. A non variable but most important disposition for special tissues are the so-called "borderline tissues", where accumulation, sedimentation and persistence of bacterial antigens are wellknown as described earlier. This phenomenon is determined by hemodynamic manifestation and quantifiably regulated by cytokines especially interleukin (IL 1) as well as by the tumor necrosis factor (TNF alpha) and prostaglandin PgE2. Additionally the cross reactivity of antibodies of Erysipelas rhusiopathiae against most specific collagen of type II, IX and XI in the pig and in laboratory animals was elaborated. This autoimmune phenomenon called "immunologic mimicry" supports besides the special physiologic conditions as niche of defense a very successful evolutionary adaptation of the agent.

Intimal asteroid bodies in horses: light and electron microscopic observations.

The morphology of asteroid bodies in equine arteries is demonstrated by light microscopy as well as by scanning and transmission electron microscopy combined with chemical analysis. Asteroid bodies first occur in horses at four weeks of age in all investigated tissues except the esophagus and always are located in the subendothelial space. The number, shape, ultrastructure and chemical composition of asteroid bodies differ markedly--depending on the age of the horse. Asteroid bodies are round and smooth in foals, but are shaped irregularly and have several projections and marked stratified calcification in adult horses. Asteroid bodies probably originate from smooth muscle cells. No direct etiological connection between asteroid bodies and migrating Strongylus vulgaris could be verified.

Borderline-tissues as sites of antigen deposition and persistence--a unifying concept of rheumatoid inflammation?

In organs of rheumatoid inflammation avascular tissue is nourished by perfusion either directly from the blood stream or by neighbouring blood vessels (borderline-tissue). An open question remains whether this borderline-tissue promotes only the known antigen persistence, or also the first deposition of the pathogenetically important microbial antigen, and subsequently a deposition of immune complexes. The three markers (carbon, latex, and living bacteria) used in this study led to nearly the same pattern of deposition in two groups of organ systems: Organs with clearance function belonging to the mononuclear phagocyte system (MPS); organs with borderline-tissues: joint, kidney, artery, heart valve, and eye. This deposition at the borderline can be observed best in areas of the joint where highly vascularized tissues are adjacent to avascular tissues nourished via perfusion: perichondrium, peritendineum, periosteum, and endo- and perimysium. These localisations of particle deposition correspond exactly with the intraarticular predilection sites of chronic rheumatoid inflammation in man and animals. Our results indicate an easier settlement of corpuscular material in these localisations, which at the same time are unable to eliminate such substances, unlike the organs of the MPS. Our studies seem to support the theory of pathogenetic importance of the borderline-tissues as a localizing factor in the perpetuating rheumatoid inflammation.

Studies of polyarthritis caused by mycoplasma arthritidis in rats. III. Histopathological findings.

Mycoplasma arthritidis induced polyarthritis in the rat is characterized histologically by four distinct phases. Firstly, two days after infection, early lesions such as vascular changes and alterations in the lining cell layer of synovium are observed. This is followed four days after inoculation by an acute exudative phase with the deposit of fibrin and infiltration by polymorphonuclear granulocytes together with first signs of joint destruction by pannus-like granulation tissue. From about the third week onwards, lymphocytes and plasma cells predominate in the subsynovium, indicating subacute inflammation. In the fourth phase which lasts until the end of the one year observation period, chronic arthritis with destruction of cartilage and bone, ankylosis, and chronic inflammation, sometimes with acute recurrences, were observed in about 25% of the joints. Both the marked tendency to pannus formation and the longevity of the arthritis, make this an interesting, experimental model of chronic polyarthritis, particularly as it has been induced by a peptidoglycan-free microorganism.

Studies of polyarthritis caused by Mycoplasma arthritidis in rats. I. Detection of the persisting Mycoplasma antigen by the enzyme immune assay (EIA) and conventional culture technique.

Investigations on Mycoplasma (M.) arthritidis polyarthritis of the rat produced by intravenous inoculation of M. arthritidis ISR 1 with special reference to the persistence of the inoculated mycoplasma antigen were performed in a total of 145 rats. The mycoplasmas were demonstrated by the conventional culture technique as well as by an enzyme immune assay (EIA). M. arthritidis was cultured from liver, kidney, spleen, and thymus up to 4 days, from trachea and uterus up to 4 weeks, and from lung, heart, brain, and lymph nodes up to 2 months after infection. It could be recovered from the joints in 70-80% of the animals up to 28 days and in the remaining 20-30% up to 200 days after infection. Using the EIA the mycoplasmal antigen could be demonstrated generally, also in the joints of infected rats which had no longer been positive by culture (10-20 weeks after inoculation). The investigations using EIA also showed a positive reaction between antiserum against M. arthritidis and joint homogenates of control rats, indicating the presence of common antigens to M. arthritidis and to joint tissues of the rat.

[Significance of vessel-free (so-called bradytrophic) tissue in the microbial colonization of model rheumatoid organs]. / Zur Bedeutung der gefässlosen (sogen. bradytrophen) Gewebe bei der mikrobiellen Besiedelung des rheumatoiden Organmusters.

The spontaneous rheumatoid disease in animals is a representative example of all the stages of rheumatism which occur in nature. It is caused exclusively by bacterial, mycoplasmal, or viral infection. The organs involved in rheumatic reactions are characterized by borders between tissue incapable of inflammation, without vessels and nourished by passive perfusion, and highly reactive, well vascularized connective tissue. By this combination microorganisms are deposited on the one hand, and then later the appearance of immune complexes is possible. These settlements are sustained by the initially occurring coagulation and permeability processes of the infection in the "vascular syndrome". After the inundation of the noxa, the bradytrophic tissue proves to be an inflammatory niche and thus an ideal antigen reservoir which can sustain the rheumatic process, in particular the humoral and the cellular immune mechanism, for the rest of the organism life. The inflammatory and immune mechanisms, probably continually induced by the persisting antigens, may not be left unconsidered during symptomatic treatment throughout the course of rheumatic diseases.

Enzyme histochemical studies of the homogeneity of the mononuclear phagocyte system with special reference to the synovium.

The cytogenetic relationship between synovial lining cell type A and macrophages both involved in joint destruction is still obscure. By means of enzyme histochemistry we tried to demonstrate marker enzymes of macrophages, like non-specific esterase and acid phosphatase in lining cells of normal and arthritic rats too. The pattern of esterase-positive cells depends on the arrangement of the lining cell layer. In flat layers single cells and in compact layers the upper row of cells react positively. The activity of acid phosphatase is negligible as compared with macrophages in liver, lung, or spleen. In arthritic joints acid phosphatase of lining cells increases and non-specific esterase concomitantly decreases. In the subsynovial tissue large amounts of esterase-positive cells occur. In these enzyme histochemical studies the pattern of marker enzymes may be indicative for a population of macrophages within the lining cell layer still under normal conditions.