The role of BMP4 signaling in trophoblast emergence from pluripotency.

The Bone Morphogenetic Protein (BMP) signaling pathway has established roles in early embryonic morphogenesis, particularly in the epiblast. More recently, however, it has also been implicated in development of extraembryonic lineages, including trophectoderm (TE), in both mouse and human. In this review, we will provide an overview of this signaling pathway, with a focus on BMP4, and its role in emergence and development of TE in both early mouse and human embryogenesis. Subsequently, we will build on these in vivo data and discuss the utility of BMP4-based protocols for in vitro conversion of primed vs. naïve pluripotent stem cells (PSC) into trophoblast, and specifically into trophoblast stem cells (TSC). PSC-derived TSC could provide an abundant, reproducible, and ethically acceptable source of cells for modeling placental development.

Early onset preeclampsia in a model for human placental trophoblast.

We describe a model for early onset preeclampsia (EOPE) that uses induced pluripotent stem cells (iPSCs) generated from umbilical cords of EOPE and control (CTL) pregnancies. These iPSCs were then converted to placental trophoblast (TB) representative of early pregnancy. Marker gene analysis indicated that both sets of cells differentiated at comparable rates. The cells were tested for parameters disturbed in EOPE, including invasive potential. Under 5% O2, CTL TB and EOPE TB lines did not differ, but, under hyperoxia (20% O2), invasiveness of EOPE TB was reduced. RNA sequencing analysis disclosed no consistent differences in expression of individual genes between EOPE TB and CTL TB under 20% O2, but, a weighted correlation network analysis revealed two gene modules (CTL4 and CTL9) that, in CTL TB, were significantly linked to extent of TB invasion. CTL9, which was positively correlated with 20% O2 (P = 0.02) and negatively correlated with invasion (P = 0.03), was enriched for gene ontology terms relating to cell adhesion and migration, angiogenesis, preeclampsia, and stress. Two EOPE TB modules, EOPE1 and EOPE2, also correlated positively and negatively, respectively, with 20% O2 conditions, but only weakly with invasion; they largely contained the same sets of genes present in modules CTL4 and CTL9. Our experiments suggest that, in EOPE, the initial step precipitating disease is a reduced capacity of placental TB to invade caused by a dysregulation of O2 response mechanisms and that EOPE is a syndrome, in which unbalanced expression of various combinations of genes affecting TB invasion provoke disease onset.

Is SARS-CoV-2 Infection a Risk Factor for Early Pregnancy Loss? ACE2 and TMPRSS2 Coexpression and Persistent Replicative Infection in Primitive Trophoblast.

BACKGROUND: SARS-CoV-2 infection in term placenta is rare. However, growing evidence suggests that susceptibility of the human placenta to infection may vary by gestational age and pathogen. For several viral infections, susceptibility appears to be greatest during early gestation. Peri-implantation placental infections that result in pre-clinical pregnancy loss would typically go undetected. Little is known about the effects of SARS-CoV-2 on the peri-implantation human placenta since this time in pregnancy can only be modeled in vitro. METHODS: We used a human embryonic stem cell (hESC)-derived model of peri-implantation placental development to assess patterns of ACE2 and TMPRSS2 transcription and protein expression in primitive trophoblast. We then infected the same trophoblast cell model with a clinical isolate of SARS-CoV-2 and documented infection dynamics. RESULTS: ACE2 and TMPRSS2 were transcribed and translated in hESC-derived trophoblast, with preferential expression in syncytialized cells. These same cells supported replicative and persistent infection by SARS-CoV-2, while non-syncytialized trophoblast cells in the same cultures did not. CONCLUSIONS: Co-expression of ACE2 and TMPRSS2 in hESC-derived trophoblast and the robust and replicative infection limited to syncytiotrophoblast equivalents support the hypothesis that increased viral susceptibility may be a defining characteristic of primitive trophoblast.

Use of a human embryonic stem cell model to discover GABRP, WFDC2, VTCN1 and ACTC1 as markers of early first trimester human trophoblast.

Human placental development during early pregnancy is poorly understood. Many conceptuses are lost at this stage. It is thought that preeclampsia, intrauterine growth restriction and other placental syndromes that manifest later in pregnancy may originate early in placentation. Thus, there is a need for models of early human placental development. Treating human embryonic stem cells (hESCs) with BMP4 (bone morphogenic protein 4) plus A83-01 (ACTIVIN/NODAL signaling inhibitor) and PD173074 (fibroblast growth factor 2 or FGF2 signaling inhibitor) (BAP conditions) induces differentiation to the trophoblast lineage (hESCBAP), but it is not clear which stage of trophoblast differentiation these cells resemble. Here, comparison of the hESCBAP transcriptome to those of trophoblasts from human blastocysts, trophoblast stem cells and placentas collected in the first-third trimester of pregnancy by principal component analysis suggests that hESC after 8 days BAP treatment most resemble first trimester syncytiotrophoblasts. To further test this hypothesis, transcripts were identified that are expressed in hESCBAP but not in cultures of trophoblasts isolated from term placentas. Proteins encoded by four genes, GABRP (gamma-aminobutyric acid type A receptor subunit Pi), WFDC2 (WAP four-disulfide core domain 2), VTCN1 (V-set domain containing T-cell activation inhibitor 1) and ACTC1 (actin alpha cardiac muscle 1), immunolocalized to placentas at 4-9 weeks gestation, and their expression declined with gestational age (R2 = 0.61-0.83). None are present at term. Expression was largely localized to syncytiotrophoblast of both hESCBAP cells and placental material from early pregnancy. WFDC2, VTCN1 and ACTC1 have not previously been described in placenta. These results support the hypothesis that hESCBAP represent human trophoblast analogous to that of early first trimester and are a tool for discovery of factors important to this stage of placentation.

Vulnerability of primitive human placental trophoblast to Zika virus.

Infection of pregnant women by Asian lineage strains of Zika virus (ZIKV) has been linked to brain abnormalities in their infants, yet it is uncertain when during pregnancy the human conceptus is most vulnerable to the virus. We have examined two models to study susceptibility of human placental trophoblast to ZIKV: cytotrophoblast and syncytiotrophoblast derived from placental villi at term and colonies of trophoblast differentiated from embryonic stem cells (ESC). The latter appear to be analogous to the primitive placenta formed during implantation. The cells from term placentas, which resist infection, do not express genes encoding most attachment factors implicated in ZIKV entry but do express many genes associated with antiviral defense. By contrast, the ESC-derived trophoblasts possess a wide range of attachment factors for ZIKV entry and lack components of a robust antiviral response system. These cells, particularly areas of syncytiotrophoblast within the colonies, quickly become infected, produce infectious virus and undergo lysis within 48 h after exposure to low titers (multiplicity of infection > 0.07) of an African lineage strain (MR766 Uganda: ZIKVU) considered to be benign with regards to effects on fetal development. Unexpectedly, lytic effects required significantly higher titers of the presumed more virulent FSS13025 Cambodia (ZIKVC). Our data suggest that the developing fetus might be most vulnerable to ZIKV early in the first trimester before a protective zone of mature villous trophoblast has been established. Additionally, MR766 is highly trophic toward primitive trophoblast, which may put the early conceptus of an infected mother at high risk for destruction.

Comparison of syncytiotrophoblast generated from human embryonic stem cells and from term placentas.

Human embryonic stem cells (ESCs) readily commit to the trophoblast lineage after exposure to bone morphogenetic protein-4 (BMP-4) and two small compounds, an activin A signaling inhibitor and a FGF2 signaling inhibitor (BMP4/A83-01/PD173074; BAP treatment). During differentiation, areas emerge within the colonies with the biochemical and morphological features of syncytiotrophoblast (STB). Relatively pure fractions of mononucleated cytotrophoblast (CTB) and larger syncytial sheets displaying the expected markers of STB can be obtained by differential filtration of dispersed colonies through nylon strainers. RNA-seq analysis of these fractions has allowed them to be compared with cytotrophoblasts isolated from term placentas before and after such cells had formed syncytia. Although it is clear from extensive gene marker analysis that both ESC- and placenta-derived syncytial cells are trophoblast, each with the potential to transport a wide range of solutes and synthesize placental hormones, their transcriptome profiles are sufficiently dissimilar to suggest that the two cell types have distinct pedigrees and represent functionally different kinds of STB. We propose that the STB generated from human ESCs represents the primitive syncytium encountered in early pregnancy soon after the human trophoblast invades into the uterine wall.

Specification of trophoblast from embryonic stem cells exposed to BMP4.

Trophoblast (TB) comprises the outer cell layers of the mammalian placenta that make direct contact with the maternal uterus and, in species with a highly invasive placenta, maternal blood. It has its origin as trophectoderm, a single epithelial layer of extra-embryonic ectoderm that surrounds the embryo proper at the blastocyst stage of development. Here, we briefly compare the features of TB specification and determination in the mouse and the human. We then review research on a model system that has been increasingly employed to study TB emergence, namely the BMP4 (bone morphogenetic protein-4)-directed differentiation of human embryonic stem cells (ESCd), and discuss why outcomes using it have proved so uneven. We also examine the controversial aspects of this model, particularly the issue of whether or not the ESCd represents TB at all. Our focus here has been to explore similarities and potential differences between the phenotypes of ESCd, trophectoderm, placental villous TB, and human TB stem cells. We then explore the role of BMP4 in the differentiation of human pluripotent cells to TB and suggest that it converts the ESC into a totipotent state that is primed for TB differentiation when self-renewal is blocked. Finally we speculate that the TB formed from ESC is homologous to the trophectoderm-derived, invasive TB that envelopes the implanting conceptus during the second week of pregnancy.

Heightened potency of human pluripotent stem cell lines created by transient BMP4 exposure.

Human pluripotent stem cells (PSCs) show epiblast-type pluripotency that is maintained with ACTIVIN/FGF2 signaling. Here, we report the acquisition of a unique stem cell phenotype by both human ES cells (hESCs) and induced pluripotent stem cells (iPSCs) in response to transient (24-36 h) exposure to bone morphogenetic protein 4 (BMP4) plus inhibitors of ACTIVIN signaling (A83-01) and FGF2 (PD173074), followed by trypsin dissociation and recovery of colonies capable of growing on a gelatin substratum in standard medium for human PSCs at low but not high FGF2 concentrations. The self-renewing cell lines stain weakly for CDX2 and strongly for NANOG, can be propagated clonally on either Matrigel or gelatin, and are morphologically distinct from human PSC progenitors on either substratum but still meet standard in vitro criteria for pluripotency. They form well-differentiated teratomas in immune-compromised mice that secrete human chorionic gonadotropin (hCG) into the host mouse and include small areas of trophoblast-like cells. The cells have a distinct transcriptome profile from the human PSCs from which they were derived (including higher expression of NANOG, LEFTY1, and LEFTY2). In nonconditioned medium lacking FGF2, the colonies spontaneously differentiated along multiple lineages, including trophoblast. They responded to PD173074 in the absence of both FGF2 and BMP4 by conversion to trophoblast, and especially syncytiotrophoblast, whereas an A83-01/PD173074 combination favored increased expression of HLA-G, a marker of extravillous trophoblast. Together, these data suggest that the cell lines exhibit totipotent potential and that BMP4 can prime human PSCs to a self-renewing alternative state permissive for trophoblast development. The results may have implications for regulation of lineage decisions in the early embryo.

Transcriptional control of IFNT expression.

Once interferon-tau (IFNT) had been identified as a type I IFN in sheep and cattle and its functions were characterized, numerous studies were conducted to elucidate the transcriptional regulation of this gene family. Transfection studies performed largely with human choriocarcinoma cell lines identified regulatory regions of the IFNT gene that appeared responsible for trophoblast-specific expression. The key finding was the recognition that the transcription factor ETS2 bound to a proximal region within the 5'UTR of a bovine IFNT and acted as a strong transactivator. Soon after other transcription factors were identified as cooperative partners. The ETS2-binding site and the nearby AP1 site enable response to intracellular signaling from maternal uterine factors. The AP1 site also serves as a GATA-binding site in one of the bovine IFNT genes. The homeobox-containing transcription factor, DLX3, augments IFNT expression combinatorially with ETS2. CDX2 has also been identified as transactivator that binds to a separate site upstream of the main ETS2 enhancer site. CDX2 participates in IFNT epigenetic regulation by modifying histone acetylation status of the gene. The IFNT downregulation at the time of the conceptus attachment to the uterine endometrium appears correlated with the increased EOMES expression and the loss of other transcription coactivators. Altogether, the studies of transcriptional control of IFNT have provided mechanistic evidence of the regulatory framework of trophoblast-specific expression and critical expression pattern for maternal recognition of pregnancy.

Engraftment of human iPS cells and allogeneic porcine cells into pigs with inactivated RAG2 and accompanying severe combined immunodeficiency.

Pigs with severe combined immunodeficiency (SCID) may provide useful models for regenerative medicine, xenotransplantation, and tumor development and will aid in developing therapies for human SCID patients. Using a reporter-guided transcription activator-like effector nuclease (TALEN) system, we generated targeted modifications of recombination activating gene (RAG) 2 in somatic cells at high efficiency, including some that affected both alleles. Somatic-cell nuclear transfer performed with the mutated cells produced pigs with RAG2 mutations without integrated exogenous DNA. Biallelically modified pigs either lacked a thymus or had one that was underdeveloped. Their splenic white pulp lacked B and T cells. Under a conventional housing environment, the biallelic RAG2 mutants manifested a "failure to thrive" phenotype, with signs of inflammation and apoptosis in the spleen compared with age-matched wild-type animals by the time they were 4 wk of age. Pigs raised in a clean environment were healthier and, following injection of human induced pluripotent stem cells (iPSCs), quickly developed mature teratomas representing all three germ layers. The pigs also tolerated grafts of allogeneic porcine trophoblast stem cells. These SCID pigs should have a variety of uses in transplantation biology.

Exploring early differentiation and pluripotency in domestic animals.

This short review describes some general features of the origins of the pluripotent inner cell mass and epiblast during the early development of eutherian mammals and the two kinds of embryonic stem cell (ESC), naïve and primed type, that have been produced from these structures. We point out that the derivation of pluripotent stem cells from domesticated species continues to be fraught with difficulties, most likely because the culture requirements of these cells are distinct from those of mouse and human ESCs. Generation of induced pluripotent stem cells (iPSCs) from the domesticated species has been more straightforward, although the majority of the iPSC lines remain dependent on the continued expression of one or more integrated reprogramming genes. Although hope for the potential usefulness of these cells in genetic modification of livestock and other domestic species has dimmed, ESCs and iPSCs remain our best source of self-renewing populations of pluripotent cells, with potential usefulness in preserving and propagating valuable animal breeds and making contributions to fields such as regenerative medicine, toxicology and even laboratory meat production.

Complete and unidirectional conversion of human embryonic stem cells to trophoblast by BMP4.

Human ES cells (hESC) exposed to bone morphogenic protein 4 (BMP4) in the absence of FGF2 have become widely used for studying trophoblast development, but the soundness of this model has been challenged by others, who concluded that differentiation was primarily toward mesoderm rather than trophoblast. Here we confirm that hESC grown under the standard conditions on a medium conditioned by mouse embryonic fibroblasts in the presence of BMP4 and absence of FGF2 on a Matrigel substratum rapidly convert to an epithelium that is largely KRT7(+) within 48 h, with minimal expression of mesoderm markers, including T (Brachyury). Instead, they begin to express a series of trophoblast markers, including HLA-G, demonstrate invasive properties that are independent of the continued presence of BMP4 in the medium, and, over time, produce extensive amounts of human chorionic gonadotropin, progesterone, placental growth factor, and placental lactogen. This process of differentiation is not dependent on conditioning of the medium by mouse embryonic fibroblasts and is accelerated in the presence of inhibitors of Activin and FGF2 signaling, which at day 2 provide colonies that are entirely KRT7(+) and in which the majority of cells are transiently CDX2(+). Colonies grown on two chemically defined media, including the one in which BMP4 was reported to drive mesoderm formation, also differentiate at least partially to trophoblast in response to BMP4. The experiments demonstrate that the in vitro BMP4/hESC model is valid for studying the emergence and differentiation of trophoblasts.

Differentiation of trophoblast cells from human embryonic stem cells: to be or not to be?

It is imperative to unveil the full range of differentiated cell types into which human pluripotent stem cells (hPSCs) can develop. The need is twofold: it will delimit the therapeutic utility of these stem cells and is necessary to place their position accurately in the developmental hierarchy of lineage potential. Accumulated evidence suggested that hPSC could develop in vitro into an extraembryonic lineage (trophoblast (TB)) that is typically inaccessible to pluripotent embryonic cells during embryogenesis. However, whether these differentiated cells are truly authentic TB has been challenged. In this debate, we present a case for and a case against TB differentiation from hPSCs. By analogy to other differentiation systems, our debate is broadly applicable, as it articulates higher and more challenging standards for judging whether a given cell type has been genuinely produced from hPSC differentiation.

The immune checkpoint molecule, VTCN1/B7-H4, guides differentiation and suppresses proinflammatory responses and MHC class I expression in an embryonic stem cell-derived model of human trophoblast.

The placenta acts as a protective barrier to pathogens and other harmful substances present in the maternal circulation throughout pregnancy. Disruption of placental development can lead to complications of pregnancy such as preeclampsia, intrauterine growth retardation and preterm birth. In previous work, we have shown that expression of the immune checkpoint regulator, B7-H4/VTCN1, is increased upon differentiation of human embryonic stem cells (hESC) to an in vitro model of primitive trophoblast (TB), that VTCN1/B7-H4 is expressed in first trimester but not term human placenta and that primitive trophoblast may be uniquely susceptible to certain pathogens. Here we report on the role of VTCN1 in trophoblast lineage development and anti-viral responses and the effects of changes in these processes on major histocompatibility complex (MHC) class I expression and peripheral NK cell phenotypes.

Large-scale production of human blastoids amenable to modeling blastocyst development and maternal-fetal cross talk.

Recent advances in human blastoids have opened new avenues for modeling early human development and implantation. One limitation of our first protocol for human blastoid generation was relatively low efficiency. We now report an optimized protocol for the efficient generation of large quantities of high-fidelity human blastoids from naive pluripotent stem cells. This enabled proteomics analysis that identified phosphosite-specific signatures potentially involved in the derivation and/or maintenance of the signaling states in human blastoids. Additionally, we uncovered endometrial stromal effects in promoting trophoblast cell survival, proliferation, and syncytialization during co-culture with blastoids and blastocysts. Side-by-side single-cell RNA sequencing revealed similarities and differences in transcriptome profiles between pre-implantation blastoids and blastocysts, as well as post-implantation cultures, and uncovered a population resembling early migratory trophoblasts during co-culture with endometrial stromal cells. Our optimized protocol will facilitate broader use of human blastoids as an accessible, perturbable, scalable, and tractable model for human blastocysts.

Leukemia inhibitory factor (LIF)-dependent, pluripotent stem cells established from inner cell mass of porcine embryos.

The pig is important for agriculture and as an animal model in human and veterinary medicine, yet despite over 20 years of effort, there has been a failure to generate pluripotent stem cells analogous to those derived from mouse embryos. Here we report the production of leukemia inhibitory factor-dependent, so-called naive type, pluripotent stem cells from the inner cell mass of porcine blastocysts by up-regulating expression of KLF4 and POU5F1. The alkaline phosphatase-positive colonies resulting from reprogramming resemble mouse embryonic stem cells in colony morphology, cell cycle interval, transcriptome profile, and expression of pluripotent markers, such as POU5F1, SOX2, and surface marker SSEA1. They are dependent on leukemia inhibitory factor signaling for maintenance of pluripotency, can be cultured over extended passage, and have the ability to form teratomas. These cells derived from the inner cell mass of pig blastocysts are clearly distinct from the FGF2-dependent "primed" induced pluripotent stem cells described recently from porcine mesenchymal cells. The data are consistent with the hypothesis that the up-regulation of KLF4, as well as POU5F1, is required to create and stabilize the naive pluripotent state and may explain why the derivation of embryonic stem cells from pigs and other ungulates has proved so difficult.

Model systems for studying trophoblast differentiation from human pluripotent stem cells.

This review focuses on a now well-established model for generating cells of the trophoblast (TB) lineage by treating human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) with the growth factor BMP4. We first discuss the opposing roles of FGF2 and BMP4 in directing TB formation and the need to exclude the former from the growth medium to minimize the co-induction of mesoderm and endoderm. Under these conditions, there is up-regulation of several transcription factors implicated in TB lineage emergence within 3 h of BMP4 exposure and, over a period of days and especially under a high O(2) gas atmosphere, gradual appearance of cell types carrying markers for more differentiated TB cell types, including extravillous TB and syncytioTB. We describe the potential value of including low molecular weight pharmaceutical agents that block activin A (INHBA) and FGF2 signaling to support BMP4-directed differentiation. We contend that the weight of available evidence supports the contention that BMP4 converts human ESC and iPSC of the so-called epiblast type unidirectionally to TB. We also consider the argument that BMP4 treatment of human ESC in the absence of exogenous FGF2 leads only to the emergence of mesoderm derivatives to be seriously flawed. Instead, we propose that, when signaling networks supporting pluripotency ESC or iPSC become unsustainable and when specification towards extra-embryonic mesoderm and endoderm are rendered inoperative, TB emerges as a major default state to pluripotency.

Differentiation of induced pluripotent stem cells of swine into rod photoreceptors and their integration into the retina.

Absence of a regenerative pathway for damaged retina following injury or disease has led to experiments using stem cell transplantation for retinal repair, and encouraging results have been obtained in rodents. The swine eye is a closer anatomical and physiological match to the human eye, but embryonic stem cells have not been isolated from pig, and photoreceptor differentiation has not been demonstrated with induced pluripotent stem cells (iPSCs) of swine. Here, we subjected iPSCs of swine to a rod photoreceptor differentiation protocol consisting of floating culture as embryoid bodies followed by differentiation in adherent culture. Real-time PCR and immunostaining of differentiated cells demonstrated loss of expression of the pluripotent genes POU5F1, NANOG, and SOX2 and induction of rod photoreceptor genes RCVRN, NRL, RHO, and ROM1. While these differentiated cells displayed neuronal morphology, culturing on a Matrigel substratum triggered a further morphological change resulting in concentration of rhodopsin (RHO) and rod outer segment-specific membrane protein 1 in outer segment-like projections resembling those on primary cultures of rod photoreceptors. The differentiated cells were transplanted into the subretinal space of pigs treated with iodoacetic acid to eliminate rod photoreceptors. Three weeks after transplantation, engrafted RHO+ cells were evident in the outer nuclear layer where photoreceptors normally reside. A portion of these transplanted cells had generated projections resembling outer segments. These results demonstrate that iPSCs of swine can differentiate into photoreceptors in culture, and these cells can integrate into the damaged swine neural retina, thus, laying a foundation for future studies using the pig as a model for retinal stem cell transplantation.