Identification of HTRA4 as a Transcriptional Target of p63 in Trophoblast.

The placenta plays a crucial role in pregnancy success. ΔNp63α (p63), a transcription factor from the TP53 family, is highly expressed in villous cytotrophoblasts (CTBs), the epithelial stem cells of the human placenta, and is involved in CTB maintenance and differentiation. We examined the mechanisms of action of p63 by identifying its downstream targets. Gene expression changes were evaluated following overexpression and knockdown of p63 in the JEG3 choriocarcinoma cell line, using microarray-based RNA profiling. High-temperature requirement A4 (HTRA4), a placenta-specific serine protease involved in trophoblast differentiation and altered in preeclampsia, was identified as a gene reciprocally regulated by p63, and its expression was characterized in primary human placental tissues by RNA-sequencing and in situ hybridization. Potential p63 DNA-binding motifs were identified in the HTRA4 promoter, and p63 occupancy at some of these sites was confirmed using chromatin immunoprecipitation, followed by quantitative PCR in both JEG3 and trophoblast stem cells. These data begin to identify members of the transcriptional network downstream of p63, thus laying the groundwork for probing mechanisms by which this important transcription factor regulates trophoblast stemness and differentiation.

Histopathologic and Transcriptomic Profiling Identifies Novel Trophoblast Defects in Patients With Preeclampsia and Maternal Vascular Malperfusion.

Preeclampsia (PE) is a heterogeneous disease for which the current clinical classification system is based on the presence or absence of specific clinical features. PE-associated placentas also show heterogeneous findings on pathologic examination, suggesting that further subclassification is possible. We combined clinical, pathologic, immunohistochemical, and transcriptomic profiling of placentas to develop integrated signatures for multiple subclasses of PE. In total, 303 PE and 1388 nonhypertensive control placentas were included. We found that maternal vascular malperfusion (MVM) in the placenta was associated with preterm PE with severe features and with small-for-gestational-age neonates. Interestingly, PE placentas with either MVM or no histologic pattern of injury showed a linear decrease in proliferative (p63+) cytotrophoblast per villous area with increasing gestational age, similar to placentas obtained from the nonhypertensive patient cohort; however, PE placentas with fetal vascular malperfusion or villitis of unknown etiology lost this phenotype. This is mainly because of cases of fetal vascular malperfusion in placentas of patients with preterm PE and villitis of unknown etiology in placentas of patients with term PE, which are associated with a decrease or increase, respectively, in the cytotrophoblast per villous area. Finally, a transcriptomic analysis identified pathways associated with hypoxia, inflammation, and reduced cell proliferation in PE-MVM placentas and further subclassified this group into extravillous trophoblast-high and extravillous trophoblast-low PE, confirmed using an immunohistochemical analysis of trophoblast lineage-specific markers. Our findings suggest that within specific histopathologic patterns of placental injury, PE can be subclassified based on specific cellular and molecular defects, allowing the identification of pathways that may be targeted for diagnostic and therapeutic purposes.

Exercise for affect and enjoyment in overweight or obese males: a comparison of high-intensity interval training and moderate-intensity continuous training.

High-intensity interval training (HIIT) is effective for generating positive cardiovascular health and fitness benefits. This study compared HIIT and moderate-intensity continuous training (MICT) for affective state and enjoyment in sedentary males with overweight or obesity.Twenty-eight participants performed stationary cycling for 6 weeks × 3 sessions/week. Participants were randomly allocated to HIIT (N=16) (10 × 1-minute intervals at ~90% peak heart rate) or MICT (N=12) (30 minutes at 65-75% peak heart rate). Affective state changes were assessed after 6-weeks training. Enjoyment and acute change in affect were assessed after individual training sessions.HIIT participants reported improved positive affect following 6 weeks training (∆ 3.6 ± 4.6, p = 0.007, effect size d = 0.70), without corresponding improvement in negative affect (p = 0.48, d = -0.19). MICT did not induce any improvement in positive affect (p = 0.56, d = 0.16) or negative affect (p = 0.23, d = -0.41). Enjoyment ratings were comparable for both exercise formats (HIIT: 4.4 ± 0.4 on a 7-point scale; MICT: 4.3 ± 0.3; p = 0.70, d = 0.15).Six weeks of HIIT induced improvement in positive affect in sedentary participants with overweight or obesity. Enjoyment of training was only slightly above neutral levels for both training formats.What's already known about this topic? Exercise training can improve general affect however the optimal exercise characteristics for improving affect are unclear.Studies assessing the relative enjoyment of HIIT in comparison to MICT have largely been equivocal to date.What does this study add? HIIT can improve affective state in males with overweight or obesity.Six weeks of stationary cycling HIIT were rated as only mildly enjoyable, comparable to ratings for MICT.

Metabolic rescue in pluripotent cells from patients with mtDNA disease.

Mitochondria have a major role in energy production via oxidative phosphorylation, which is dependent on the expression of critical genes encoded by mitochondrial (mt)DNA. Mutations in mtDNA can cause fatal or severely debilitating disorders with limited treatment options. Clinical manifestations vary based on mutation type and heteroplasmy (that is, the relative levels of mutant and wild-type mtDNA within each cell). Here we generated genetically corrected pluripotent stem cells (PSCs) from patients with mtDNA disease. Multiple induced pluripotent stem (iPS) cell lines were derived from patients with common heteroplasmic mutations including 3243A>G, causing mitochondrial encephalomyopathy and stroke-like episodes (MELAS), and 8993T>G and 13513G>A, implicated in Leigh syndrome. Isogenic MELAS and Leigh syndrome iPS cell lines were generated containing exclusively wild-type or mutant mtDNA through spontaneous segregation of heteroplasmic mtDNA in proliferating fibroblasts. Furthermore, somatic cell nuclear transfer (SCNT) enabled replacement of mutant mtDNA from homoplasmic 8993T>G fibroblasts to generate corrected Leigh-NT1 PSCs. Although Leigh-NT1 PSCs contained donor oocyte wild-type mtDNA (human haplotype D4a) that differed from Leigh syndrome patient haplotype (F1a) at a total of 47 nucleotide sites, Leigh-NT1 cells displayed transcriptomic profiles similar to those in embryo-derived PSCs carrying wild-type mtDNA, indicative of normal nuclear-to-mitochondrial interactions. Moreover, genetically rescued patient PSCs displayed normal metabolic function compared to impaired oxygen consumption and ATP production observed in mutant cells. We conclude that both reprogramming approaches offer complementary strategies for derivation of PSCs containing exclusively wild-type mtDNA, through spontaneous segregation of heteroplasmic mtDNA in individual iPS cell lines or mitochondrial replacement by SCNT in homoplasmic mtDNA-based disease.

Abnormalities in human pluripotent cells due to reprogramming mechanisms.

Human pluripotent stem cells hold potential for regenerative medicine, but available cell types have significant limitations. Although embryonic stem cells (ES cells) from in vitro fertilized embryos (IVF ES cells) represent the 'gold standard', they are allogeneic to patients. Autologous induced pluripotent stem cells (iPS cells) are prone to epigenetic and transcriptional aberrations. To determine whether such abnormalities are intrinsic to somatic cell reprogramming or secondary to the reprogramming method, genetically matched sets of human IVF ES cells, iPS cells and nuclear transfer ES cells (NT ES cells) derived by somatic cell nuclear transfer (SCNT) were subjected to genome-wide analyses. Both NT ES cells and iPS cells derived from the same somatic cells contained comparable numbers of de novo copy number variations. In contrast, DNA methylation and transcriptome profiles of NT ES cells corresponded closely to those of IVF ES cells, whereas iPS cells differed and retained residual DNA methylation patterns typical of parental somatic cells. Thus, human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal for cell replacement therapies.

Concise Review: Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer: A Horse in the Race?

Embryonic stem cells (ESC) hold promise for the treatment of human medical conditions but are allogeneic. Here, we consider the differences between autologous pluripotent stem cells produced by nuclear transfer (NT-ESCs) and transcription factor-mediated, induced pluripotent stem cells (iPSCs) that impact the desirability of each of these cell types for clinical use. The derivation of NT-ESCs is more cumbersome and requires donor oocytes; however, the use of oocyte cytoplasm as the source of reprogramming factors is linked to a key advantage of NT-ESCs-the ability to replace mutant mitochondrial DNA in a patient cell (due to either age or inherited disease) with healthy donor mitochondria from an oocyte. Moreover, in epigenomic and transcriptomic comparisons between isogenic iPSCs and NT-ESCs, the latter produced cells that more closely resemble bona fide ESCs derived from fertilized embryos. Thus, although NT-ESCs are more difficult to generate than iPSCs, the ability of somatic cell nuclear transfer to replace aged or diseased mitochondria and the closer epigenomic and transcriptomic similarity between NT-ESCs and bona fide ESCs may make NT-ESCs superior for future applications in regenerative medicine. Stem Cells 2017;35:26-34.

Spontaneous Single-Copy Loss of TP53 in Human Embryonic Stem Cells Markedly Increases Cell Proliferation and Survival.

Genomic aberrations have been identified in many human pluripotent stem cell (hPSC) cultures. Commonly observed duplications in portions of chromosomes 12p and 17q have been associated with increases in genetic instability and resistance to apoptosis, respectively. However, the phenotypic consequences related to sporadic mutations have not been evaluated to date. Here, we report on the effects of a single-copy deletion of the chr17p13.1 region, a sporadic mutation that spontaneously arose independently in several subclones of a human embryonic stem cell culture. Compared to cells with two normal copies of chr17p13.1 ("wild-type"), the cells with a single-copy deletion of this region ("mutant") displayed a selective advantage when exposed to stressful conditions, and retained a higher percentage of cells expressing the pluripotency marker POU5F1/OCT4 after 2 weeks of in vitro differentiation. Knockdown of TP53, which is a gene encompassed by the deleted region, in wild-type cells mimicked the chr17p13.1 deletion phenotype. Thus, sporadic mutations in hPSCs can have phenotypic effects that may impact their utility for clinical applications. Stem Cells 2017;35:872-885.

An extended wave of global mRNA deadenylation sets up a switch in translation regulation across the mammalian oocyte-to-embryo transition.

Without new transcription, gene expression across the oocyte-to-embryo transition (OET) relies instead on regulation of mRNA poly(A) tails to control translation. However, how tail dynamics shape translation across the OET in mammals remains unclear. We perform long-read RNA sequencing to uncover poly(A) tail lengths across the mouse OET and, incorporating published ribosome profiling data, provide an integrated, transcriptome-wide analysis of poly(A) tails and translation across the entire transition. We uncover an extended wave of global deadenylation during fertilization in which short-tailed, oocyte-deposited mRNAs are translationally activated without polyadenylation through resistance to deadenylation. Subsequently, in the embryo, mRNAs are readenylated and translated in a surge of global polyadenylation. We further identify regulation of poly(A) tail length at the isoform level and stage-specific enrichment of mRNA sequence motifs among regulated transcripts. These data provide insight into the stage-specific mechanisms of poly(A) tail regulation that orchestrate gene expression from oocyte to embryo in mammals.

Molecular profiling of human blastocysts reveals primitive endoderm defects among embryos of decreased implantation potential.

Human embryo implantation is remarkably inefficient, and implantation failure remains among the greatest obstacles in treating infertility. Gene expression data from human embryos have accumulated rapidly in recent years; however, identification of the subset of genes that determine successful implantation remains a challenge. We leverage clinical morphologic grading-known for decades to correlate with implantation potential-and transcriptome analyses of matched embryonic and abembryonic samples to identify factors and pathways enriched and depleted in human blastocysts of good and poor morphology. Unexpectedly, we discovered that the greatest difference was in the state of extraembryonic primitive endoderm (PrE) development, with relative deficiencies in poor morphology blastocysts. Our results suggest that implantation success is most strongly influenced by the embryonic compartment and that deficient PrE development is common among embryos with decreased implantation potential. Our study provides a valuable resource for those investigating the markers and mechanisms of human embryo implantation.

iPSC-based modeling of preeclampsia identifies epigenetic defects in extravillous trophoblast differentiation.

Preeclampsia (PE) is a hypertensive pregnancy disorder with increased risk of maternal and fetal morbidity and mortality. Abnormal extravillous trophoblast (EVT) development and function is considered to be the underlying cause of PE, but has not been previously modeled in vitro. We previously derived induced pluripotent stem cells (iPSCs) from placentas of PE patients and characterized abnormalities in formation of syncytiotrophoblast and responses to changes in oxygen tension. In this study, we converted these primed iPSC to naïve iPSC, and then derived trophoblast stem cells (TSCs) and EVT to evaluate molecular mechanisms underlying PE. We found that primed (but not naïve) iPSC-derived PE-EVT have reduced surface HLA-G, blunted invasive capacity, and altered EVT-specific gene expression. These abnormalities correlated with promoter hypermethylation of genes associated with the epithelial-mesenchymal transition pathway, specifically in primed-iPSC derived PE-EVT. Our findings indicate that abnormal epigenetic regulation might play a role in PE pathogenesis.

Microbes, metagenomes and marine mammals: enabling the next generation of scientist to enter the genomic era.

BACKGROUND: The revolution in DNA sequencing technology continues unabated, and is affecting all aspects of the biological and medical sciences. The training and recruitment of the next generation of researchers who are able to use and exploit the new technology is severely lacking and potentially negatively influencing research and development efforts to advance genome biology. Here we present a cross-disciplinary course that provides undergraduate students with practical experience in running a next generation sequencing instrument through to the analysis and annotation of the generated DNA sequences. RESULTS: Many labs across world are installing next generation sequencing technology and we show that the undergraduate students produce quality sequence data and were excited to participate in cutting edge research. The students conducted the work flow from DNA extraction, library preparation, running the sequencing instrument, to the extraction and analysis of the data. They sequenced microbes, metagenomes, and a marine mammal, the Californian sea lion, Zalophus californianus. The students met sequencing quality controls, had no detectable contamination in the targeted DNA sequences, provided publication quality data, and became part of an international collaboration to investigate carcinomas in carnivores. CONCLUSIONS: Students learned important skills for their future education and career opportunities, and a perceived increase in students' ability to conduct independent scientific research was measured. DNA sequencing is rapidly expanding in the life sciences. Teaching undergraduates to use the latest technology to sequence genomic DNA ensures they are ready to meet the challenges of the genomic era and allows them to participate in annotating the tree of life.

Modeling placental development and disease using human pluripotent stem cells.

Our current knowledge of the cellular and molecular mechanisms of placental epithelial cells, trophoblast, primarily came from the use of mouse trophoblast stem cells and tumor-derived or immortalized human trophoblast cell lines. This was mainly due to the difficulties in maintaining primary trophoblast in culture and establishing human trophoblast stem cell (hTSC) lines. However, in-depth characterization of these cellular models and in vivo human trophoblast have revealed significant discrepancies. For the past two decades, multiple groups have shown that human pluripotent stem cells (hPSCs) can be differentiated into trophoblast, and thus could be used as a model for normal and disease trophoblast differentiation. During this time, trophoblast differentiation protocols have evolved, enabling researchers to study cellular characteristics at trophectoderm (TE), trophoblast stem cells (TSC), syncytiotrophoblast (STB), and extravillous trophoblast (EVT) stages. Recently, several groups reported methods to derive hTSC from pre-implantation blastocyst or early gestation placenta, and trophoblast organoids from early gestation placenta, drastically changing the landscape of trophoblast research. These culture conditions have been rapidly applied to generate hPSC-derived TSC and trophoblast organoids. As a result of these technological advancements, the field's capacity to better understand trophoblast differentiation and their involvement in pregnancy related disease has greatly expanded. Here, we present in vitro models of human trophoblast differentiation, describing both primary and hPSC-derived TSC, maintained as monolayers and 3-dimensional trophoblast organoids, as a tool to study early placental development and disease in multiple settings.

Activation of the Interferon Pathway in Trophoblast Cells Productively Infected with SARS-CoV-2.

SARS-CoV-2 infection during pregnancy has been associated with poor maternal and neonatal outcomes and placental defects. The placenta, which acts as a physical and immunological barrier at the maternal-fetal interface, is not established until the end of the first trimester. Therefore, localized viral infection of the trophoblast compartment early in gestation could trigger an inflammatory response resulting in altered placental function and consequent suboptimal conditions for fetal growth and development. In this study, we investigated the effect of SARS-CoV-2 infection in early gestation placentae using placenta-derived human trophoblast stem cells (TSCs), a novel in vitro model, and their extravillous trophoblast (EVT) and syncytiotrophoblast (STB) derivatives. SARS-CoV-2 was able to productively replicate in TSC-derived STB and EVT, but not undifferentiated TSCs, which is consistent with the expression of SARS-CoV-2 entry host factors, ACE2 (angiotensin-converting enzyme 2) and TMPRSS2 (transmembrane cellular serine protease) in these cells. In addition, both TSC-derived EVT and STB infected with SARS-CoV-2 elicited an interferon-mediated innate immune response. Combined, these results suggest that placenta-derived TSCs are a robust in vitro model to investigate the effect of SARS-CoV-2 infection in the trophoblast compartment of the early placenta and that SARS-CoV-2 infection in early gestation activates the innate immune response and inflammation pathways. Therefore, placental development could be adversely affected by early SARS-CoV-2 infection by directly infecting the developing differentiated trophoblast compartment, posing a higher risk for poor pregnancy outcomes.

Corrigendum: Transcriptomic drivers of differentiation, maturation, and polyploidy in human extravillous trophoblast.

[This corrects the article DOI: 10.3389/fcell.2021.702046.].

High placental expression of FLT1, LEP, PHYHIP and IL3RA - In persons of African ancestry with severe preeclampsia.

INTRODUCTION: Mortality from preeclampsia (PE) and PE-associated morbidities are 3-to 5-fold higher in persons of African ancestry than in those of Asian and European ancestries. METHODS: To elucidate placental contribution to worse PE outcomes in African ancestry pregnancies, we performed bulk RNA sequencing on 50 placentas from persons with severe PE (sPE) of African (n = 9), Asian (n = 18) and European (n = 23) ancestries and 73 normotensive controls of African (n = 10), Asian (n = 15) and European (n = 48) ancestries. RESULTS: Previously described canonical preeclampsia genes, involved in metabolism and hypoxia/angiogenesis including: LEP, HK2, FSTL3, FLT1, ENG, TMEM45A, ARHGEF4 and HTRA1 were upregulated sPE versus normotensive placentas across ancestries. LTF, NPR3 and PHYHIP were higher in African vs. Asian ancestry sPE placentas. Allograft rejection/adaptive immune response genes were upregulated in placentas from African but not in Asian or European ancestry sPE patients; IL3RA was of particular interest because the patient with the highest placental IL3RA expression, a person of African ancestry with sPE, developed postpartum cardiomyopathy, and was the only patient out of 123, that developed this condition. Interestingly, the sPE patients with the highest IL3RA expression among persons of Asian and European ancestries developed unexplained tachycardia peripartum, necessitating echocardiography in the European ancestry patient. The association between elevated placental IL3RA levels and unexplained tachycardia or peripartum cardiomyopathy was found to be significant in the 50 sPE patients (p = .0005). DISCUSSION: High placental upregulation of both canonical preeclampsia and allograft rejection/adaptive immune response genes may contribute to worse PE outcomes in African ancestry sPE patients.

Discovery and verification of extracellular microRNA biomarkers for diagnostic and prognostic assessment of preeclampsia at triage.

We report on the identification of extracellular miRNA (ex-miRNA) biomarkers for early diagnosis and prognosis of preeclampsia (PE). Small RNA sequencing of maternal serum prospectively collected from participants undergoing evaluation for suspected PE revealed distinct patterns of ex-miRNA expression among different categories of hypertensive disorders in pregnancy. Applying an iterative machine learning method identified three bivariate miRNA biomarkers (miR-522-3p/miR-4732-5p, miR-516a-5p/miR-144-3p, and miR-27b-3p/let-7b-5p) that, when applied serially, distinguished between PE cases of different severity and differentiated cases from controls with a sensitivity of 93%, specificity of 79%, positive predictive value (PPV) of 55%, and negative predictive value (NPV) of 89%. In a small independent validation cohort, these ex-miRNA biomarkers had a sensitivity of 91% and specificity of 57%. Combining these ex-miRNA biomarkers with the established sFlt1:PlGF protein biomarker ratio performed better than either set of biomarkers alone (sensitivity of 89.4%, specificity of 91.3%, PPV of 95.5%, and NPV of 80.8%).

Stabilization of hESCs in two distinct substates along the continuum of pluripotency.

A detailed understanding of the developmental substates of human pluripotent stem cells (hPSCs) is needed to optimize their use in cell therapy and for modeling early development. Genetic instability and risk of tumorigenicity of primed hPSCs are well documented, but a systematic isogenic comparison between substates has not been performed. We derived four hESC lines in naive human stem cell medium (NHSM) and generated isogenic pairs of NHSM and primed cultures. Through phenotypic, transcriptomic, and methylation profiling, we identified changes that arose during the transition to a primed substate. Although early NHSM cultures displayed naive characteristics, including greater proliferation and clonogenic potential compared with primed cultures, they drifted toward a more primed-like substate over time, including accumulation of genetic abnormalities. Overall, we show that transcriptomic and epigenomic profiling can be used to place human pluripotent cultures along a developmental continuum and may inform their utility for clinical and research applications.

Derivation of functional trophoblast stem cells from primed human pluripotent stem cells.

Trophoblast stem cells (TSCs) have recently been derived from human embryos and early-first-trimester placenta; however, aside from ethical challenges, the unknown disease potential of these cells limits their scientific utility. We have previously established a bone morphogetic protein 4 (BMP4)-based two-step protocol for differentiation of primed human pluripotent stem cells (hPSCs) into functional trophoblasts; however, those trophoblasts could not be maintained in a self-renewing TSC-like state. Here, we use the first step from this protocol, followed by a switch to newly developed TSC medium, to derive bona fide TSCs. We show that these cells resemble placenta- and naive hPSC-derived TSCs, based on their transcriptome as well as their in vitro and in vivo differentiation potential. We conclude that primed hPSCs can be used to generate functional TSCs through a simple protocol, which can be applied to a widely available set of existing hPSCs, including induced pluripotent stem cells, derived from patients with known birth outcomes.

Transcriptomic Drivers of Differentiation, Maturation, and Polyploidy in Human Extravillous Trophoblast.

During pregnancy, conceptus-derived extravillous trophoblast (EVT) invades the endomyometrium, anchors the placenta to the maternal uterus, and remodels the spiral arteries in order to establish maternal blood supply to the fetoplacental unit. Recent reports have described early gestation EVT as polyploid and senescent. Here, we extend these reports by performing comprehensive profiling of both the genomic organization and transcriptome of first trimester and term EVT. We define pathways and gene regulatory networks involved in both initial differentiation and maturation of this important trophoblast lineage at the maternal-fetal interface. Our results suggest that like first trimester EVT, term EVT undergoes senescence and endoreduplication, is primarily tetraploid, and lacks high rates of copy number variations. Additionally, we have highlighted senescence and polyploidy-related genes, pathways, networks, and transcription factors that appeared to be important in normal EVT differentiation and maturation and validated a key role for the unfolded protein response in this context.

Modeling preeclampsia using human induced pluripotent stem cells.

Preeclampsia (PE) is a pregnancy-specific hypertensive disorder, affecting up to 10% of pregnancies worldwide. The primary etiology is considered to be abnormal development and function of placental cells called trophoblasts. We previously developed a two-step protocol for differentiation of human pluripotent stem cells, first into cytotrophoblast (CTB) progenitor-like cells, and then into both syncytiotrophoblast (STB)- and extravillous trophoblast (EVT)-like cells, and showed that it can model both normal and abnormal trophoblast differentiation. We have now applied this protocol to induced pluripotent stem cells (iPSC) derived from placentas of pregnancies with or without PE. While there were no differences in CTB induction or EVT formation, PE-iPSC-derived trophoblast showed a defect in syncytialization, as well as a blunted response to hypoxia. RNAseq analysis showed defects in STB formation and response to hypoxia; however, DNA methylation changes were minimal, corresponding only to changes in response to hypoxia. Overall, PE-iPSC recapitulated multiple defects associated with placental dysfunction, including a lack of response to decreased oxygen tension. This emphasizes the importance of the maternal microenvironment in normal placentation, and highlights potential pathways that can be targeted for diagnosis or therapy, while absence of marked DNA methylation changes suggests that other regulatory mechanisms mediate these alterations.