Mana M. Parast

Recurrent Variations in DNA Methylation in Human Pluripotent Stem Cells and Their Differentiated Derivatives

Human pluripotent stem cells (hPSCs) are potential sources of cells for modeling disease and development, drug discovery, and regenerative medicine. However, it is important to identify factors that may impact the utility of hPSCs for these applications. In an unbiased analysis of 205 hPSC and 130 somatic samples, we identified hPSC-specific epigenetic and transcriptional aberrations in genes subject to X chromosome inactivation (XCI) and genomic imprinting, which were not corrected during directed differentiation. We also found that specific tissue types were distinguished by unique patterns of DNA hypomethylation, which were recapitulated by DNA demethylation during in vitro directed differentiation. Our results suggest that verification of baseline epigenetic status is critical for hPSC-based disease models in which the observed phenotype depends on proper XCI or imprinting and that tissue-specific DNA methylation patterns can be accurately modeled during directed differentiation of hPSCs, even in the presence of variations in XCI or imprinting.

Serous Tubal Intraepithelial Carcinoma: Its Potential Role in Primary Peritoneal Serous Carcinoma and Serous Cancer Prevention

PURPOSE A diagnosis of primary peritoneal serous carcinoma (PPSC) requires exclusion of a source in other reproductive organs. Serous tubal intraepithelial carcinoma (STIC; stage 0) has been described in asymptomatic women with BRCA mutations and linked to a serous cancer precursor in the fimbria. This study examined the frequency of STIC in PPSC and its clinical outcome in BRCA-positive women. PATIENTS AND METHODS Presence or absence of STIC was recorded in consecutive cases meeting the 2001 WHO criteria for PPSC, including 26 patients with nonuniform sampling of the fallopian tubes (group 1) and 19 patients with complete tubal examination (group 2; sectioning and extensively examining the fimbriated end, or SEE-FIM protocol). In selected cases, STIC or its putative precursor and the peritoneal tumor were analyzed for p53 mutations (exons 1 to 11). Outcome of STIC was ascertained by literature review. RESULT Thirteen (50%) of 26 PPSCs in group 1 involved the endosalpinx, with nine STICs (35%). Fifteen (79%) of 19 cases in group 2 contained endosalpingeal involvement, with nine STICs (47%). STIC was typically fimbrial and unifocal, with variable invasion of the tubal wall. In five of five cases, the peritoneal and tubal lesion shared an identical p53 mutation. Of 10 reported STICs in BRCA-positive women, all patients were without disease on follow-up. CONCLUSION The fimbria is the source of nearly one half of PPSCs, suggesting serous malignancy originates in the tubal mucosa but grows preferentially at a remote peritoneal site. The generally low risk of recurrence in stage 0 (STIC) disease further underscores STIC as a possible target for early serous cancer detection and prevention.

Pathophysiology of Placenta Creta: The Role of Decidua and Extravillous Trophoblast

Placenta creta is associated with massive postpartum hemorrhage and commonly leads to emergency hysterectomy. While the exact pathogenesis of placenta creta is unknown, proposed hypotheses include a primary deficiency of decidua, abnormal maternal vascular remodeling, excessive trophoblastic invasion, or a combination thereof. To assess these changes in placenta creta, we retrospectively reviewed 49 cases of gravid hysterectomy, 38 with and 11 without the diagnosis of creta, gathered clinical data, and evaluated histopathology of extravillous trophoblast. Specifically, we evaluated maternal vessels for remodeling by endovascular trophoblast, as well as the morphology and depth of invasion of interstitial trophoblast at the implantation site. Compared to controls, cases with creta had decreased proportion of remodeled vessels, with many vessels displaying partial physiologic change. Cases with creta also demonstrated vascular remodeling deeper in the myometrium; however, vascular remodeling of large outer myometrial vessels was only demonstrated in increta and percreta cases, and was absent in both non-creta and accreta. As previously reported, interstitial trophoblast invaded the uterine wall to a significantly greater depth in placenta creta; however, there was no significant difference between creta subtypes. Finally, Ki-67 staining was rarely observed in extravillous trophoblast, except in the trophoblast columns of first trimester creta cases. We, therefore, conclude that the pathogenesis of placenta creta is multi-dimensional, involving increased, but incomplete trophoblast invasion in a background of absent decidua. We further propose that placenta increta and percreta are not due to a further invasion of extravillous trophoblast in the uterine wall, rather they likely arise secondary to dehiscence of a scar, leading to the presence of chorionic villi deep within the uterine wall, and thus give extravillous trophoblast greater access to the deep myometrium.

Statistically based splicing detection reveals neural enrichment and tissue-specific induction of circular RNA during human fetal development.

BackgroundThe pervasive expression of circular RNA is a recently discovered feature of gene expression in highly diverged eukaryotes, but the functions of most circular RNAs are still unknown. Computational methods to discover and quantify circular RNA are essential. Moreover, discovering biological contexts where circular RNAs are regulated will shed light on potential functional roles they may play.ResultsWe present a new algorithm that increases the sensitivity and specificity of circular RNA detection by discovering and quantifying circular and linear RNA splicing events at both annotated and un-annotated exon boundaries, including intergenic regions of the genome, with high statistical confidence. Unlike approaches that rely on read count and exon homology to determine confidence in prediction of circular RNA expression, our algorithm uses a statistical approach. Using our algorithm, we unveiled striking induction of general and tissue-specific circular RNAs, including in the heart and lung, during human fetal development. We discover regions of the human fetal brain, such as the frontal cortex, with marked enrichment for genes where circular RNA isoforms are dominant.ConclusionsThe vast majority of circular RNA production occurs at major spliceosome splice sites; however, we find the first examples of developmentally induced circular RNAs processed by the minor spliceosome, and an enriched propensity of minor spliceosome donors to splice into circular RNA at un-annotated, rather than annotated, exons. Together, these results suggest a potentially significant role for circular RNA in human development.

Specific requirement for two ADF/cofilin isoforms in distinct actin-dependent processes in Caenorhabditis elegans

Actin depolymerizing factor (ADF)/cofilin is an essential enhancer of actin turnover. Multicellular organisms express multiple ADF/cofilin isoforms in different patterns of tissue distribution. However, the functional significance of different ADF/cofilin isoforms is not understood. The Caenorhabditis elegans unc-60 gene generates two ADF/cofilins, UNC-60A and UNC-60B, by alternative splicing. These two ADF/cofilin proteins have different effects on actin dynamics in vitro, but their functional difference in vivo remains unclear. Here, we demonstrate that the two isoforms are expressed in different tissues and are required for distinct morphogenetic processes. UNC-60A was ubiquitously expressed in most embryonic cells and enriched in adult gonads, intestine and oocytes. In contrast, UNC-60B was specifically expressed in the body wall muscle, vulva and spermatheca. RNA interference of UNC-60A caused embryonic lethality with variable defects in cytokinesis and developmental patterning. In severely affected embryos, a cleavage furrow was formed and progressed but reversed before completion of the cleavage. Also, in some affected embryos, positioning of the blastomeres became abnormal, which resulted in embryonic arrest. In contrast, an unc-60B-null mutant was homozygous viable, underwent normal early embryogenesis and caused disorganization of actin filaments specifically in body wall muscle. These results suggest that the ADF/cofilin isoforms play distinct roles in specific aspects of actin reorganization in vivo.

Gross Abnormalities of the Umbilical Cord: Related Placental Histology and Clinical Significance

OBJECTIVE To evaluate umbilical cord abnormalities predisposing to mechanical cord compression and determine their relationship to adverse clinical outcomes and stasis-associated histologic changes in the placenta. METHODS Placental slides of 224 singleton pregnancies with gross cord abnormality (true knots, long cords, nuchal/body cords, abnormal cord insertion, hypercoiled cords, narrow cords with diminished Whartons jelly), delivered on or after 28 weeks gestational age, and 317 gestational age-matched controls, were reviewed and specifically evaluated for the following histologic changes: (1) fetal vascular ectasia, (2) fetal vascular thrombosis, (3) and fetal thrombotic vasculopathy/avascular villi. These changes were analyzed in relation to both clinical information and findings at gross pathologic examination. RESULTS Gross cord abnormalities were associated with stillbirth, intrauterine growth restriction, non-reassuring fetal tracing, meconium-stained amniotic fluid, and increased rate of emergency Cesarean section. At microscopic evaluation, cases with gross cord abnormalities showed a statistically significant association with both ectasia and thrombosis in the fetal vasculature, as well as changes of fetal thrombotic vasculopathy in the terminal villi. When considering individual gross cord abnormalities, long cord and nuchal cord had the highest rates of thrombosis-related histopathology. Finally, cases with both abnormal cords and histologic thrombosis had significantly higher rates of adverse outcomes, including IUGR and stillbirth. CONCLUSION Gross cord abnormalities predispose the fetus to stasis-induced vascular ectasia and thrombosis, thus leading to vascular obstruction and adverse neonatal outcome, including IUGR and stillbirth. We recommend a thorough histopathologic evaluation of all placentas with gross cord abnormalities predisposing to cord compression.

Role of astroglia in Down’s syndrome revealed by patient-derived human-induced pluripotent stem cells

Down’s syndrome (DS), caused by trisomy of human chromosome 21, is the most common genetic cause of intellectual disability. Here we use induced pluripotent stem cells (iPSCs) derived from DS patients to identify a role for astrocytes in DS pathogenesis. DS astroglia exhibit higher levels of reactive oxygen species and lower levels of synaptogenic molecules. Astrocyte-conditioned medium collected from DS astroglia causes toxicity to neurons, and fails to promote neuronal ion channel maturation and synapse formation. Transplantation studies show that DS astroglia do not promote neurogenesis of endogenous neural stem cells in vivo. We also observed abnormal gene expression profiles from DS astroglia. Finally, we show that the FDA-approved antibiotic drug, minocycline, partially corrects the pathological phenotypes of DS astroglia by specifically modulating the expression of S100B, GFAP, inducible nitric oxide synthase, and thrombospondins 1 and 2 in DS astroglia. Our studies shed light on the pathogenesis and possible treatment of DS by targeting astrocytes with a clinically available drug. Down’s syndrome is characterized by intellectual disability and other neuropathological symptoms. Here, the authors show that astroglia derived from induced pluripotent stem cells from Down’s syndrome patients impair the development of neurons, and that this can be attenuated with the drug minocycline.

Siglec-5 and Siglec-14 are polymorphic paired receptors that modulate neutrophil and amnion signaling responses to group B Streptococcus

Siglec-5 and Siglec-14 are shown to be paired inhibitory/activating receptors expressed on neutrophils and amniotic epithelium and modulating immune responses to group B Streptococcus.

The proline-rich protein palladin is a binding partner for profilin

Palladin is an actin‐associated protein that has been suggested to play critical roles in establishing cell morphology and maintaining cytoskeletal organization in a wide variety of cell types. Palladin has been shown previously to bind directly to three different actin‐binding proteins vasodilator‐stimulated phosphoprotein (VASP), α‐actinin and ezrin, suggesting that it functions as an organizing unit that recruits actin‐regulatory proteins to specific subcellular sites. Palladin contains sequences resembling a motif known to bind profilin. Here, we demonstrate that palladin is a binding partner for profilin, interacting with profilin via a poly proline‐containing sequence in the amino‐terminal half of palladin. Double‐label immunofluorescence staining shows that palladin and profilin partially colocalize in actin‐rich structures in cultured astrocytes. Our results suggest that palladin may play an important role in recruiting profilin to sites of actin dynamics.

Obstetric and Perinatal Complications in Placentas with Fetal Thrombotic Vasculopathy

Fetal thrombotic vasculopathy (FTV) is a placental lesion characterized by regionally distributed avascular villi and is often accompanied by upstream thrombosis in placental fetal vessels. Previous studies, using preselected populations, have shown associations of this lesion with adverse neurodevelopmental outcomes and potentially obstructive lesions of the umbilical cord. We investigated the prevalence of obstetric complications, perinatal disease, and placental abnormalities in cases with FTV. One hundred thirteen cases of placentas with FTV were identified in our pathology database over an 18-year period. Two hundred sixteen placentas without the diagnosis of FTV, frequency matched on year of birth, were selected as controls. Electronic medical records and pathology reports were used to extract maternal and gestational age, method of delivery, neonatal outcome, lesions of the umbilical cord, obstetric complications, and fetal abnormalities. Placentas with FTV were associated with a 9-fold increase in rate of stillbirth and a 2-fold increase in intrauterine growth restriction. The increase in pregnancy-induced hypertension/preeclampsia was not significant when adjusted for maternal and gestational age. Although the rate of potentially obstructive cord lesions was similar in both groups, there was an almost 6-fold increase in the presence of oligohydramnios in FTV placentas, compared with controls. Finally, FTV was associated with a 6-fold increase in fetal cardiac abnormalities. Fetal thrombotic vasculopathy is associated with a significantly higher rate of obstetric and perinatal complications. This study points to abnormal fetal circulation, either in the form of congenital heart disease or oligohydramnios predisposing to cord compression, as a risk factor for FTV.