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.

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.

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.

Beyond fusion: A novel role for ERVW-1 in trophoblast proliferation and type I interferon receptor expression.

INTRODUCTION: Throughout human pregnancy there is a delicate balance between the maintenance of a proliferative, trophoblast stem cell pool (TSC) and the differentiation from TSC to placental cell sub-lineages like the syncytiotrophoblast (STB). The STB is comprised of multinucleated cells that come into direct contact with maternal blood and provides the first line of defense to protect the fetus from maternal infections. The differentiation of TSC towards STB is primarily driven by human endogenous retroviruses (HERV), specifically Syncytin-1 (ERVW-1) and Syncytin-2 (ERVFRD-1). Beyond cell fusion, there is also evidence to suggest they can regulate cell proliferation and an antiviral response in other cell types. Therefore, we hypothesized that HERV can regulate cell proliferation as well as an antiviral response in TSCs. METHOD: shRNA was used to knockdown ERVW-1 in TSCs and revealed reduction in cell proliferation, differentiation, and cell fusion. RT-qPCR and flow cytometry was used to measure other HERV and the presence of Type I and Type II interferon receptors. RESULTS: ERVW-1 knockdown (KD) TSCs had a significantly longer cell doubling time and reduced expression of the proliferation marker Ki67. ERVW-1 KD cells also demonstrated a marked deficiency in the ability to differentiate. Interestingly, ERVFRD-1 was upregulated in both ERVW-1 KD TSC and STB cells compared to controls. Finally, we found that the Type I interferon receptors, IFNAR1 and IFNAR2 were significantly increased in ERVW-1 KD STB cells. DISCUSSION: These findings uncover critical HERV functions in the trophoblasts and a novel role for ERVW-1 during early human placental development.

The product of BMP-directed differentiation protocols for human primed pluripotent stem cells is placental trophoblast and not amnion.

The observation that trophoblast (TB) can be generated from primed pluripotent stem cells (PSCs) by exposure to bone morphogenetic protein-4 (BMP4) when FGF2 and ACTIVIN signaling is minimized has recently been challenged with the suggestion that the procedure instead produces amnion. Here, by analyzing transcriptome data from multiple sources, including bulk and single-cell data, we show that the BMP4 procedure generates bona fide TB with similarities to both placental villous TB and TB generated from TB stem cells. The analyses also suggest that the transcriptomic signatures between embryonic amnion and different forms of TB have commonalities. Our data provide justification for the continued use of TB derived from PSCs as a model for investigating placental development.

Single Nucleus RNA Sequence (snRNAseq) Analysis of the Spectrum of Trophoblast Lineages Generated From Human Pluripotent Stem Cells in vitro.

One model to study the emergence of the human trophoblast (TB) has been the exposure of pluripotent stem cells to bone morphogenetic protein 4 (BMP4) in presence of inhibitors of ACTIVIN/TGFB; A83-01 and FGF2; PD173074 (BAP), which generates a mixture of cytotrophoblast, syncytiotrophoblast, and cells with similarities to extravillous trophoblast. Here, H1 human embryonic stem cells were BAP-exposed under two O2 conditions (20% and 5%, respectively). At day 8, single nuclei RNA sequencing was used for transcriptomics analysis, thereby allowing profiling of fragile syncytial structures as well as the more resilient mononucleated cells. Following cluster analysis, two major groupings, one comprised of five (2,4,6,7,8) and the second of three (1,3,5) clusters were evident, all of which displayed recognized TB markers. Of these, two (2 and 3) weakly resembled extravillous trophoblast, two (5 and 6) strongly carried the hallmark transcripts of syncytiotrophoblast, while the remaining five were likely different kinds of mononucleated cytotrophoblast. We suggest that the two populations of nuclei within syncytiotrophoblast may have arisen from fusion events involving two distinct species of precursor cells. The number of differentially expressed genes between O2 conditions varied among the clusters, and the number of genes upregulated in cells cultured under 5% O2 was highest in syncytiotrophoblast cluster 6. In summary, the BAP model reveals an unexpectedly complex picture of trophoblast lineage emergence that will need to be resolved further in time-course studies.

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.

Transcriptome analysis of MBD5-associated neurodevelopmental disorder (MAND) neural progenitor cells reveals dysregulation of autism-associated genes.

MBD5-associated neurodevelopmental disorder (MAND) is an autism spectrum disorder (ASD) characterized by intellectual disability, motor delay, speech impairment and behavioral problems; however, the biological role of methyl-CpG-binding domain 5, MBD5, in neurodevelopment and ASD remains largely undefined. Hence, we created neural progenitor cells (NPC) derived from individuals with chromosome 2q23.1 deletion and conducted RNA-seq to identify differentially expressed genes (DEGs) and the biological processes and pathways altered in MAND. Primary skin fibroblasts from three unrelated individuals with MAND and four unrelated controls were converted into induced pluripotent stem cell (iPSC) lines, followed by directed differentiation of iPSC to NPC. Transcriptome analysis of MAND NPC revealed 468 DEGs (q < 0.05), including 20 ASD-associated genes. Comparison of DEGs in MAND with SFARI syndromic autism genes revealed a striking significant overlap in biological processes commonly altered in neurodevelopmental phenotypes, with TGFß, Hippo signaling, DNA replication, and cell cycle among the top enriched pathways. Overall, these transcriptome deviations provide potential connections to the overlapping neurocognitive and neuropsychiatric phenotypes associated with key high-risk ASD genes, including chromatin modifiers and epigenetic modulators, that play significant roles in these disease states.

Syncytins expressed in human placental trophoblast.

Three versions of syncytiotrophoblast exist in the human placenta: an invasive type associated with the implanting conceptus, non-invasive villous type of definitive placenta, and placental bed giant cells. Syncytins are encoded by modified env genes of endogenous retroviruses (ERV), but how they contribute functionally to placental syncytial structures is unclear. A minimum of eight genes (ERVW1, ERVFRD-1, ERVV-1, ERVV-2, ERVH48-1, ERVMER34-1, ERV3-1, & ERVK13-1) encoding syncytin family members are expressed in human trophoblast, the majority from implantation to term. ERVW1 (Syncytin 1) and ERVFRD-1 (Syncytin 2) are considered the major fusogens, but, when the expression of their genes is analyzed by single cell RNAseq in first trimester placenta, their transcripts are distinctly patterned and also differ from those of their proposed binding partners, SLC1A5 and MFSD2A, respectively. ERVRH48-1 (suppressyn or SUPYN) and ERVMER34-1 are probable negative regulators of fusion and co-expressed, primarily in cytotrophoblast. The remaining genes and their products have been little studied. Syncytin expression is a feature of placental development in almost all eutherian mammals studied, in at least one marsupial, and in viviparous lizards, which lack the trophoblast lineage. Their expression has been inferred to be essential for pregnancy success in the mouse. All the main human ERV genes arose following independent retroviral insertion events, none of which trace back to the divergence of eutherians and metatherians (marsupials). While syncytins may be crucial for placental development, it seems unlikely that they helped orchestrate the divergence of eutherians and marsupials.

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.

A six-inhibitor culture medium for improving naïve-type pluripotency of porcine pluripotent stem cells.

Understanding essential signaling network requirements and making appropriate adjustments in culture conditions are crucial if porcine pluripotent stem cells (PSC) are to achieve their full potential. Here, we first used two protein factors (LIF and FGF2) and kinase inhibitor combinations in attempts to convert primed type lentiviral-reprogrammed porcine induced PSC (Lv-piPSC) into naïve-like state and developed a medium called FL6i. In addition to FGF2 and LIF, this medium contained inhibitors of MAPK14, MAPK8, TGFB1, MAP2K1, GSK3A and BMP. Crucially, the usual TGFB1 and BMP4 protein components of many stem cell media were replaced in FL6i with inhibitors of TGFB1 and BMP. With this medium, Lv-piPSC were readily transformed from their original primed state into cells that formed colonies with typical features of naïve-state stem cells. The FL6i medium also assisted generation of naïve-type piPSC lines from porcine embryonic fibroblasts with non-integrating episomal plasmids (Epi-piPSC). These lines, despite retaining variable amounts of vector DNA, expressed higher endogenous pPOU5F1 and pSOX2 than Lv-piPSC. They have been cultured without obvious morphological change for >45 passages and retained pluripotent phenotypes in terms of upregulation of genes associated with pluripotency, low expression of genes linked to emergence of somatic cell lineages, and ability to generate well differentiated teratomas in immune-compromised mice. FL6i conditions, therefore, appear to support elevated pluripotent phenotypes. However, FL6i was less able to support the generation of embryonic stem cells from porcine blastocysts. Although colonies with dome-shaped morphologies were evident and the cells had some gene expression features linked to pluripotency, the phenotypes were ultimately not stable. Pathway analysis derived from RNAseq data performed on the various cell lines generated in this study suggest the benefits of employing the FL6i medium on porcine cells reside in its ability to minimize TGFB1 and BMP signaling, which would otherwise de-stabilize the stem cell state.

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.

African and Asian strains of Zika virus differ in their ability to infect and lyse primitive human placental trophoblast.

Zika virus (ZIKV) drew worldwide attention when a recent epidemic was linked to fetal microcephaly. Here we used human embryonic stem cell derived trophoblasts as a model for primitive placental trophoblast to test the hypothesis that there are differences in how the two genetically distinct ZIKV lineages, African (AF) and Asian (AS), target the human placenta. Upon infection with three AF (ib-H30656, SEN/1984/41525-DAK, and MR-766) and three AS (FSS13025, MexI-44, and PANcdc259249) ZIKV strains, we observed that severe placental cell lysis was only induced after infection with AF strains, while viral replication rates remained similar between both lineages. Differences in cytopathic effects (CPE) were not observed in Vero cells, indicating that the AF strains were not inherently superior at cell lysis. Taken together, we propose that infection with AF strains of ZIKV early in pregnancy would likely result in pregnancy loss, rather than allow further fetal development with accompanying brain damage. Our results also suggest that the long term laboratory-adapted MR-766 strain does not behave aberrantly in cell culture relative to other AF lineage strains.

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.

Deciphering transcriptional regulation in human embryonic stem cells specified towards a trophoblast fate.

Differentiated human embryonic stem cells (hESC) continue to provide a model for studying early trophoblast cells (TB), but many questions have been raised regarding their true identity. Therefore, we carried out a global and unbiased analysis on previously published transcriptomic profiles for hESC differentiated to TB by means of bone morphogenetic protein-4 and inhibitors of activin A and fibroblast growth factor-2 signaling (BAP treatment). Our results confirm that BAP treated hESC (ESCd) lack a mesoderm signature and are a subtype of placental cells unlike those present at term. ESCd display a high level of expression of genes implicated in migration and invasion compared to commonly used, immortalized TB cell lines and primary cells from term placenta. Co-expression network analysis also identified gene modules involved in cell migration and adhesion, processes that are likely critical during the beginning stages of placentation. Finally, protein-protein interaction analysis predicted several additional genes that may play important roles in early stages of placental development. Together, our analyses provide novel insights into the transcriptional programs that are active in ESCd.

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.

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.