Normal Fetal Liver Research Articles

Prenatal quantification of human foetal lung and liver elasticities between 24 and 39 weeks of gestation using 2D shear wave elastography

ObjectivesTo quantify and model normal foetal lung and liver elasticities between 24 and 39 weeks of gestation (WG) using two-dimensional shear wave elastography (2D-SWE). To assess the impact of the distance between the probe and the target organ on the estimation of elasticity values.MethodsMeasurements of normal foetal lungs and liver elasticity were prospectively repeated monthly between 24 and 39 WG in 72 foetuses using 2D-SWE. Elasticity was quantified in the proximal lung and in the region inside the hepatic portal sinus. The distance between the probe and the target organ was recorded. Trajectories representing foetal lung and liver maturation from at least 3 measurements over time were modelled.ResultsThe average elasticity for the lung and liver was significantly different from 24 WG to 36 WG (p < 0.01). Liver elasticity increased during gestation (3.86 kPa at 24 WG versus 4.45 kPa at 39 WG). From 24 WG to 32 WG, lung elasticity gradually increased (4.12kPa at 24 WG, 4.91kPa at 28 WG, 5.03kPa at 32 WG, p < 0.002). After 32 WG, lung elasticity decreased to 4.54kPa at 36 WG and 3.94kPa at 39 WG. The dispersion of the average elasticity values was greater for the lung than for the liver (p < 0.0001). Variation in the elasticity values was less important for the liver than for the lung. The values were considered valid and repeatable except for a probe-lung distance above 8cm.ConclusionFoetal lung and liver elasticities evolve differently through gestation. This could reflect the tissue maturation of both organs during gestation.Trial registrationclinicaltrials.gov identifier: NCT03834805Key Points• Prenatal quantification of foetal lung elasticity using 2D shear wave elastography could be a new prenatal parameter for exploring foetal lung maturity.• Liver elasticity increased progressively from 24 weeks of gestation (WG) to 39 WG, while lung elasticity increased first between 24 and 32 WG and then decreased after 32 WG.• The values of elasticity are considered valid and repeatable except for a probe-lung distance above 8cm.

A Human Model of Down Syndrome Associated Leukemia Reveals Different Cell of Origins for Initiation and Progression

Introduction: Leukemia is the most common cancer in children and sequencing data suggest that the first genetic alterations often occur in utero. Children with Down syndrome (Trisomy 21, T21) have a 150-fold increased risk of childhood leukemia. In 30% of newborns with Down syndrome, a transient myeloproliferative disorder (pre-leukemia) occurs, which is characterized by a clonal proliferation of immature megakaryoblasts carrying somatic mutations in the GATA1 transcription factor (GATA1s) and resolves spontaneously in most cases. In 20% of the cases, acute megakaryoblastic leukemia (AMKL) evolves from the pre-leukemic clone by acquisition of additional mutations, such as in the cohesin subunit STAG2. It is hypothesized that this represents a multi-step process of leukemogenesis with three distinct genetic events: T21, GATA1s and STAG2. Yet, it remains unclear how an extra copy of chromosome 21 predisposes towards leukemia, the interplay between each genetic event and the cellular origin of transformation. Methods: Human long-term hematopoietic stem cells (LT-HSCs) were sorted from normal karyotype and T21 fetal livers (N-FL and T21-FL) and subsequently CRISPR/Cas9 edited to try to establish a humanized model of Down Syndrome associated pre-leukemia and AMKL. To model the initiation of the pre-leukemic state, GATA1s mutations were introduced, while additional STAG2- mutations were overlaid to model the progression to fully transformed AMKL. CRISPR/Cas9-edited control, GATA1s, STAG2- and GATA1s/STAG2- LT-HSCs were functionally interrogated in near-clonal xenograft assays, along with transcriptional and epigenetic profiling. Results: T21 status in combination with GATA1s had a profound synergistic effect on megakaryocytic lineage output in vivo compared to normal karyotype with GATA1s. Moreover, a high percentage of blasts were found in xenografts of GATA1s edited T21-FL LT-HSCs (&amp;gt;30%) but not in xenografts of GATA1s edited N-FL LT-HSCs. Conversely, GATA1s/STAG2- edited LT-HSCs generated grafts with &amp;gt;50% of blasts, regardless of T21 status. The immunophenotype of these blasts recapitulated those observed in patients diagnosed with Down Syndrome pre-leukemia and AMKL (CD117+CD34+CD41+CD71+CD33+CD4+CD7+). Thus, T21 is required for pre-leukemia development, but seems dispensable for AMKL as both N- and T21-FL LT-HSCs underwent leukemic transformation upon GATA1s/STAG2-. Serial xenotransplantation assays from primary engrafted mice were carried out to assess self-renewal properties of GATA1s-induced pre-leukemia and GATA1s/STAG2- induced AMKL. Only GATA1s/STAG2- edited N- and T21-FL grafts were able to propagate the leukemic phenotype with a high stem cell frequency, which was endowed by the additional STAG2- knock-out. ATACseq and RNAseq profiling of blast populations revealed an enrichment of GATA-binding sites with concomitant up-regulation of genes implicated in translation. To assess the role of progenitor cells in pre-leukemic initiation and leukemic progression, we CRISPR/Cas9 edited short-term HSCs, common myeloid progenitors and myelo-erythroid progenitors with GATA1s and/or STAG2- and subjected them to xenotransplantation. Strikingly, all progenitor subsets with combined GATA1s/STAG2- editing were able to drive leukemic transformation, while single GATA1s editing in the same subsets did not initiate pre-leukemia. This data strongly suggests that the initial GATA1s mutation must occur in T21 LT-HSCs, but subsequent STAG2 mutations can occur further downstream in progenitors. Lastly, to gain insight into how chromosome 21 predisposes towards pre-leukemia, three chromosome 21 miRNAs (miR-99a, -125b-2 and -155) were identified to be up-regulated in T21-FL LT-HSCs compared to N-FL LT-HSCs. Over-expression of these miRNAs in N-FL LT-HSCs induced a T21-like state with increased myeloid and megakaryocytic skewing. Dramatically, CRISPR/Cas9-edited knock-out of these miRNAs in GATA1s edited T21-FL LT-HSCs resulted in a block of pre-leukemia initiation. Conclusion: Our findings demonstrate that T21 is required for pre-leukemia initiation, which is mediated by over-expression of chromosome 21 miRNAs in LT-HSCs. Further, this data demonstrates different cell of origins between pre-leukemia initiation and AMKL progression. Ongoing studies focus on preventing the progression of pre-leukemia to AMKL by pharmacological targeting. Figure Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding.

Understanding Pre-Leukemia in Trisomy 21 Human HSC and Modeling Progression Towards Down Syndrome Associated Leukemia Using CRISPR/Cas9 at Single Cell Resolution

Leukemia is the most common cancer in children. Sequencing data from identical twins suggests that the first genetic alterations in childhood leukemia occur in utero. Children with Down syndrome (Trisomy 21, T21) have an increased risk of childhood leukemia. In 30% of newborns with Down syndrome, a transient pre-leukemia disease occurs, which is characterized by a clonal proliferation of immature megakaryocytes carrying somatic mutations in the GATA1 transcription factor. These acquired GATA1 mutations lead to the expression of an N-terminal truncated protein (GATA1-Short). In 20% of the cases, acute megakaryoblastic leukemia (AMKL) evolves from the pre-leukemia by acquisition of additional genetic mutations in the transient leukemia clone, predominantly in genes of the cohesin complex. It is hypothesized that this represents a multi-step process of leukemogenesis with three distinct genetic events: T21, GATA1-Short and additional cohesin mutations. Yet, it remains unclear how an extra copy of chromosome 21 predisposes towards leukemia, the mechanisms of leukemic transformation and the interplay between each genetic component. Therefore, we wanted to establish a tractable human model system to investigate the initiation and evolution of transient leukemia and AMKL using CRISPR/Cas9 genome editing in primary human hematopoietic stem cells (HSCs). To model the initiation of Down syndrome associated pre-leukemia, we utilized both neonatal cord blood and fetal liver derived LT-HSCs and other progenitor populations to express either the short or long isoform of GATA1 (GATA1-Short or GATA1-Long). This was carried out using an improved methodology that permits the in vitro and in vivo functional interrogation of CRISPR/Cas9 edited human LT-HSCs at the single cell level (Wagenblast et al., bioRxiv 609040). Importantly, in this case, expression of either GATA1 isoform remained under the regulatory control of the endogenous promoter. Culture of single LT-HSC, short-term (ST-HSC) and myelo-erythroid progenitors (MEP) revealed a drastic shift towards megakaryocytic lineage output upon exclusive expression of GATA1-Short compared to control or GATA1-Long, regardless of the developmental source of the derived cells. To investigate the functional consequences of exclusive GATA1-Short expression in LT-HSCs in vivo, we performed near-clonal xenotransplantation assays in NSG and NSGW41 mice. Strikingly, GATA1-Short edited LT-HSCs injected mice displayed a higher percentage of human CD41+CD45- megakaryocytic lineage derived cells and a decrease in human GlyA+CD45- erythroid cells compared to control. Morphological analysis revealed more immature forms of erythroid cells and fewer enucleated erythrocytes in GATA1-Short edited LT-HSCs injected mice. In order to add an additional genetic determinant to our model, we utilized T21 fetal liver derived LT-HSCs. Un-manipulated T21 LT-HSCs and other progenitor populations showed a bias towards erythroid, myeloid and megakaryocytic lineages at the expense of lymphoid fates. In vitro, the combination of T21 and CRISPR/Cas9-mediated GATA1-Short in LT-HSCs led to an increase in megakaryocytic lineage output, while decreasing erythroid output. This phenotype was similar to what was observed in normal karyotype fetal liver derived LT-HSCs. However, near clonal transplantation of GATA1-Short edited T21 LT-HSCs in NSG mice generated exclusive CD33+ myeloid grafts with disproportionate high levels of CD41+CD45- megakaryocytic lineage derived cells compared to T21 control. In addition a distinct CD34+CD41+CD71+CD45+ population was present. Thus, this phenotype is reminiscent of Down Syndrome associated transient leukemia. In summary, by using an improved CRISPR/Cas9 single cell methodology we show how GATA1 regulates lineage fate in normal and T21 LT-HSCs and other progenitor populations. Importantly, we show for the first time a humanized mouse model of Down syndrome associated transient leukemia, which was induced from T21 human fetal liver derived LT-HSCs engineered to express GATA1-Short. Current studies focus on adding additional mutations of the cohesin complex to progress transient leukemia to AMKL. Disclosures No relevant conflicts of interest to declare.

Bclaf1 Promotes Maintenance and Self-Renewal of Fetal Hematopoietic Stem Cells

Hematopoietic stem cells (HSC) persist throughout life by undergoing extensive self-renewal divisions while maintaining an undifferentiated state. The mechanisms that support HSC self-renewal change throughout the course of development as temporal changes in transcriptional regulators coordinate distinct genetic programs in fetal, post-natal and adult HSCs. These self-renewal programs are often ectopically activated in leukemia cells to drive neoplastic proliferation and high expression of HSC-associated genes predicts a poor prognosis in acute myelogenous leukemia (AML). In this regard, it was recently shown that expression of the transcriptional regulator BCLAF1 (Bcl2 associated transcription factor 1) is increased in AML blasts relative to normal precursor populations and suppression of BCLAF1 causes reduced proliferation and induction of differentiation to a dendritic cell fate. These findings raise the question of whether BCLAF1 may regulate normal as well as neoplastic self-renewal programs.We find that Bclaf1 is highly expressed in HSCs versus committed bone marrow populations consistent with a potential role for this gene in HSC functions. To test whether BCLAF1 regulates HSC development and hematopoiesis, we used germline loss of function mice. Bclaf1-/- mice succumb to pulmonary failure shortly after birth due to poor lung development, so we assessed prenatal hematopoiesis. Bclaf1-deficient mice had significantly reduced HSC and hematopoietic progenitor cell (HPC) frequencies and numbers despite normal fetal liver cellularity. To determine if Bclaf1 is required for HSC function during fetal development, we performed competitive reconstitution assays. Fetal liver cells from Bclaf1+/+ or Bclaf1-/- mice were transplanted along with wild-type competitor bone marrow cells into lethally irradiated recipient mice. Compared to recipients of Bclaf1+/+ fetal liver cells, recipients of Bclaf1-/- cells had a significantly lower percentage of donor-derived leukocytes at all time points after transplantation as well as reduced percentage of donor HSCs at 16 weeks post-transplant. Notably, all leukocyte populations (B cells, T cells, granulocytes and macrophages) from Bclaf1-/- donors were reduced consistent with an abnormality in HSC repopulating activity rather than a defect in a specific differentiation pathway. Consistent with these findings, Bclaf-deficient cells did not engraft in competitive transplants with limiting numbers of sorted fetal liver HSCs whereas sorted wild-type Bclaf1+/+ cells effectively reconstituted hematopoiesis in recipient mice. In addition, Vav-cre:Bclaf1flox/flox mice, which have selective deletion of Bclaf1 in hematopoietic cells, have reduced frequencies and numbers of fetal liver HSCs identical to the findings observed in germline Bclaf1-/- mice. These results show that loss of Bclaf1 leads to defective development and repopulating activity of fetal HSCs.Interestingly, when adult mice are successfully engrafted with Bclaf1-deficient HSCs, the donor HSCs suffer no additional functional impairment. Furthermore, in secondary transplant experiments Bclaf1-deficient HSCs maintain long-term repopulating activity. Thus, Bclaf1 may have distinct functions in fetal versus adult HSC self-renewal. Collectively, our findings reveal Bclaf1 is a novel regulator of fetal HSC function and suggest that it may have distinct functions in different developmental contexts. DisclosuresNo relevant conflicts of interest to declare.

FETAL LIVER MORPHOMETRY AT DIFFERENT CROWN RUMP LENGTHS

Introduction: Normal morphometric parameters of liver in human fetus were studied at various stage of development with an aim to get insight into the normal development of fetal liver. Material &amp; Methods: Dissection of 29 human fetuses was performed. Anthropometric study was done and various parameters were measured. Results: The development of liver is associated with increase in all the parameters although the increase is highly variable in different gestational age, indicating that liver development showed characteristic but highly regulated development to reach its fully developed stage. Conclusion: The present study will be helpful in understanding the normal fetal liver development.

Cell-extrinsic hematopoietic impact of Ezh2 inactivation in fetal liver endothelial cells

AbstractDespite the well-established cell-intrinsic role of epigenetic factors in normal and malignant hematopoiesis, their cell-extrinsic role remains largely unexplored. Herein we investigated the hematopoietic impact of inactivating Ezh2, a key component of polycomb repressive complex 2 (PRC2), in the fetal liver (FL) vascular niche. Hematopoietic specific (Vav-iCre) Ezh2 inactivation enhanced FL hematopoietic stem cell (HSC) expansion with normal FL erythropoiesis. In contrast, endothelium (Tie2-Cre) targeted Ezh2 inactivation resulted in embryonic lethality with severe anemia at embryonic day 13.5 despite normal emergence of functional HSCs. Ezh2-deficient FL endothelium overexpressed Mmp9, which cell-extrinsically depleted the membrane-bound form of Kit ligand (mKitL), an essential hematopoietic cytokine, in FL. Furthermore, Mmp9 inhibition in vitro restored mKitL expression along with the erythropoiesis supporting capacity of FL endothelial cells. These data establish that Ezh2 is intrinsically dispensable for FL HSCs and provides proof of principle that modulation of epigenetic regulators in niche components can exert a marked cell-extrinsic impact.

Maternal-Fetal Disposition and Metabolism of Retrorsine in Pregnant Rats.

Pyrrolizidine alkaloids (PAs) are extensively synthesized by plants, are commonly present in herbs and foodstuffs, and exhibit hepatotoxicity requiring metabolic activation by cytochrome P450 3A to form the electrophilic metabolites-pyrrolic esters. PAs also cause embryo toxicity, but the metabolic profiles of PAs in fetus and placenta have been far from clear. In this study, we determined the basal metabolic activation of retrorsine (RTS) in rat maternal liver, placenta, and fetal liver in vitro and examined the fetal toxicity and bioactivation of RTS in vivo. Detection of microsomal RTS metabolites in vitro showed that the basal metabolic activity of fetal liver and placenta to RTS was much weaker than that of maternal liver. In addition, a higher rate of pyrrolic ester formation was found in normal male fetal liver compared with that of female pups. In vivo exposure to RTS caused fetal growth retardation, as well as placental and fetal liver injury. Little difference in serum RTS was observed in dams and fetuses, but the content of pyrrole-protein adduction in the fetal liver was much lower than that in maternal liver, which was consistent with basal metabolic activity. Unexpectedly, compared with basal metabolism in fetal liver, exposure to RTS during middle and late pregnancy caused an opposite gender difference in RTS metabolism and CYP3A expression in the fetal liver. For the first time, our study showed that RTS can permeate the placenta barrier and entering fetal circulation, whereas the intrauterine pyrrolic metabolite was generated mainly by fetal liver but not transported from the maternal circulation. Induction of CYP3A by RTS was gender-dependent in the fetal liver, which was probably responsible for RTS-induced fetal hepatic injury, especially for female pups.

Fetal Erythropoiesis Is Defective in Rpl11 Heterozygous Mice and Increases in Severity in Young Animals

Diamond Blackfan Anemia (DBA) is a congenital red blood cell aplasia usually caused by haploinsufficiency of selected ribosomal protein genes. Approximately 10% of DBA patients are anemic at birth, and 80% of DBA patients present by 6 months. These and other data suggest that the DBA erythroid defect is exacerbated by the switch from fetal to adult hematopoiesis. We hypothesize that unique molecular or cellular features initiate DBA pathogenesis during fetal hematopoiesis that manifests after the switch to adult erythropoiesis as severe anemia.To test this hypothesis, we developed a mouse model with constitutive Rpl11 haploinsufficiency in fetal and adult hematopoietic tissues driven by VavCre. Rpl11 is a commonly mutated ribosomal gene in DBA. In adult mice, reduced Rpl11 expression results in severe anemia by 6 weeks, with markedly reduced hemoglobin, increased red cell volume and elevated erythroid adenosine deaminase. Flow cytometric analysis of the bone marrow (BM) revealed no change in the frequency of megakaryocyte-erythroid progenitors (MEP) but a significant increase in both BFU-E and CFU-E, with a corresponding decrease in maturing Ter119+ cells. Functional progenitor assays showed reduced and delayed proliferation with an impaired ability to produce viable CD71+ Ter119+ cells. Treatment of purified Rpl11+/- BFU-E with dexamethasone rescued this proliferative defect. Thus, our data indicate that a differentiation block and functional impairment at the BFU-E stage of adult erythropoiesis contribute to Rpl11 haploinsufficient anemia.At the molecular level, Rpl11 haploinsufficiency in BM resulted in the accumulation of 32S and 12S pre-rRNA. Polysome profiling of total BM suggested reduced polysome assembly and impaired translation. Transcriptional profiling of normal and Rpl11+/- MEP, BFU-E, CFU-E and Ter119+ cells identified 13 differentially expressed genes across DBA erythropoiesis including known and novel targets such as CDKN1A, TNFSF4 and TSPAN14 . Enrichment of the p53 pathway was unique to BFU-E and CFU-E progenitors. Interestingly, TGFB1 and EGF pathways were also selectively affected within Rpl11+/- BFU-E and CFU-E progenitors, suggesting p53-independent self-renewal and proliferative defects in these populations.In contrast to adult hematopoiesis, fetal hematopoiesis defects are less severe. For example, fetal BFU-E are not significantly increased within E14 fetal livers and functionally generate a similar number of maturing erythroblasts in vitro . Lodish and colleagues have established that fetal liver BFU-E comprise at least two distinct subpopulations, termed “early” and “late”. Early BFU-E have high self-renewing capacity whereas late BFU-E have low self-renewing capacity. To determine whether the balance between early and late BFU-E is affected by Rpl11 haploinsufficiency, and to identify gene expression changes in early and late fetal Rpl11+/- BFU-E, we performed single cell RNA-sequencing.Normal fetal livers contained similar proportions of early and late BFU-E, whereas normal adult bone marrows only contained late BFU-E. Surprisingly, Rpl11+/- fetal livers primarily contained late BFU-E. One explanation for this observation is that early BFU-E are depleted from Rpl11+/- fetal livers as this population compensates for reduced function of non-renewing late BFU-E. Interestingly, late BFU-E from Rpl11+/- BM have a stronger “late” signature than their normal counterparts suggesting a reduced self-renewing capacity of BM BFU-E in DBA. We are currently testing this hypothesis by overexpressing early genes associated with BFU-E self-renewal in Rpl11+/- BFU-E and reassessing erythroid phenotypes.In conclusion, our studies show that erythropoiesis is defective in Rpl11+/- fetal liver at the cellular and molecular level, although functional impairment is not as marked as in adult mice. Moreover, single cell RNA-sequencing of BM and fetal BFU-E in our Rpl11+/- mouse model has uncovered a novel hypothesis for DBA pathogenesis that the loss of early, self-renewing BFU-E in fetal liver is a harbinger of adult erythroid failure. DisclosuresGlader:Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Neuberg:Synta Pharmaceuticals: Other: Stock shares.

Tetrandrine antagonizes acute megakaryoblastic leukaemia growth by forcing autophagy-mediated differentiation.

The poor prognosis of acute megakaryoblastic leukaemia (AMKL) means there is a need to develop novel therapeutic methods to treat this condition. It was recently shown that inducing megakaryoblasts to undergo terminal differentiation is effective as a treatment for AMKL. This encouraged us to identify a compound that induces megakaryocyte differentiation, which could then act as a potent anti-leukaemia agent. The effects of tetrandrine on the expression of CD41 and cell morphology were investigated in AMKL cells. We used CRISPR/Cas9 knockout system to knock out ATG7 and verify the role of autophagy in tetrandrine-induced megakaryocyte differentiation. shNotch1 and CA-Akt were transfected into K562 cells to examine the downstream pathways of ROS signalling and the mechanistic basis of the tetrandrine-induced megakaryocyte differentiation. The anti-leukaemia effects of tetrandrine were analysed both in vitro and in vivo. A low dose of tetrandrine induced cell cycle arrest and megakaryocyte differentiation in AMKL cells via activation of autophagy. Molecularly, we demonstrated that this effect is mediated by activation of Notch1 and Akt and subsequent accumulation of ROS. In contrast, in normal mouse fetal liver cells, although tetrandrine induced autophagy, it did not affect cell proliferation or promote megakaryocyte differentiation, suggesting a specific effect of tetrandrine in malignant megakaryoblasts. Finally, tetrandrine also showed in vivo efficacy in an AMKL xenograft mouse model. Modulating autophagy-mediated differentiation may be a novel strategy for treating AMKL, and tetrandrine has the potential to be developed as a differentiation-inducing agent for AMKL chemotherapy.

PORTACAVAL FIBROUS CONNECTIONS: LITTLE KNOWN ANATOMICAL STRUCTURES OF LIVER

Background &amp; Aims. Studies dealing with the architecture of the portal and caval canals of liver have overlooked the probability of the fibrous connections between the major portal complexes and hepatic veins when they touch each other. Such a probability is essential for both theoretical and practical medicine. Methods. We studied macro- and micro-morphological specimens from 104 normal adult and 18 normal embryonic and fetal livers. Results. We found that the fibrous sheathes of the portal complexes and hepatic veins fuse in certain areas where these structures cross each other. This anatomical relationship between the portal tracts and hepatic veins is a normal occurrence in human liver and originates at 11th-12th week of embryogenesis. Conclusions. The paper is an effort to demonstrate anatomical and clinical significance and embryogenesis of the portacaval fibrous connections.

Comparative Cytotoxicity and Sperm Motility Using a Computer-Aided Sperm Analysis System (CASA) for Isomers of Phthalic Acid, a Common Final Metabolite of Phthalates

The general population is exposed to phthalates through consumer products, diet, and medical devices. Phthalic acid (PA) is a common final metabolite of phthalates, and its isomers include isophthalic acid (IPA), terephthalic acid (TPA), and phthalaldehyde (o-phthalic acid, OPA). The purpose of this study was to investigate whether PA and PA isomers exert reproductive toxicity, including altered sperm movement. In vitro cell viability assays were comparatively performed using Sertoli and liver cell lines. In animal experiments, PA or PA isomers (10, 100, or 1000 mg/kg) were administered orally to Sprague-Dawley (SD) rats, and semen samples were analyzed by computer-aided sperm analysis (CASA). PA treatment produced a significant effect on curvilinear velocity (VCL), straight-line velocity (VSL), mean velocity or average path velocity (VAP), amplitude of lateral head displacement (ALH), and frequency of head displacement or beat cross-frequency (BCF), whereas IPA, TPA, and OPA induced no marked effects. In vitro cell viability assays showed that mouse normal testis cells (TM4) and human testis cancer cells (NTERA 2 cl. D1) were more sensitive to PA and OPA than mouse liver normal cells (NCTC clone 1469) and human fetal liver cells (FL 62891). Our study suggests that PA and PA isomers specifically produced significant in vitro and in vivo reproductive toxicity, particularly sperm toxicity and testis cell cytotoxicity. Of the isomers examined, PA appeared to be the most toxic and may serve as a surrogate biomarker for reproductive toxicity following mixed exposure to phthalates.

Deubiquitinase MYSM1 Is Essential for Normal Fetal Liver Hematopoiesis and for the Maintenance of Hematopoietic Stem Cells in Adult Bone Marrow.

MYSM1 is a chromatin-interacting deubiquitinase recently shown to be essential for hematopoietic stem cell (HSC) function and normal progression of hematopoiesis in both mice and humans. However, it remains unknown whether the loss of function in Mysm1-deficient HSCs is due to the essential role of MYSM1 in establishing the HSC pool during development or due to a continuous requirement for MYSM1 in adult HSCs. In this study we, for the first time, address these questions first, by performing a detailed analysis of hematopoiesis in the fetal livers of Mysm1-knockout mice, and second, by assessing the effects of an inducible Mysm1 ablation on adult HSC functions. Our data indicate that MYSM1 is essential for normal HSC function and progression of hematopoiesis in the fetal liver. Furthermore, the inducible knockout model demonstrates a continuous requirement for MYSM1 to maintain HSC functions and antagonize p53 activation in adult bone marrow. These studies advance our understanding of the role of MYSM1 in HSC biology, and provide new insights into the human hematopoietic failure syndrome resulting from MYSM1 deficiency.

A study on histology of fetal liver

Background and aims : The liver during fetogenesis does not follow classical lobular pattern. Normal histology of the fetal liver at various stages of development was studied to get insight into the morphology of fetal liver and special function it performs in fetal life. Method: Dissection of 29 normal human fetuses was done and histological findings of liver were noted with respect to the age of fetus. The histology of fetal liver was studied using H & E stain and important features of fetal liver were studied. Result: Fetal liver histology is different from adult liver. Unlike adult liver, fetal liver shows hepatocytes arranged in sheets predominantly with cordlike pattern at places. The sinusoids are dilated and filled with hemopoietic cells, which could be appreciated at different stages of fetal development. The Kupffer cells were also noticed in fetal liver. Conclusion: The present study will be helpful in understanding the normal histology of fetal liver and add to the existing knowledge regarding development of fetal liver.

Nutlin-3-induced redistribution of chromatin-bound IFI16 in human hepatocellular carcinoma cells in vitro is associated with p53 activation.

Interferon-γ inducible protein 16 (IFI16), a DNA sensor for DNA double-strand break (DSB), is expressed in most human hepatocellular carcinoma cell (HCC) lines. In this study we investigated the re-localization of chromatin-bound IFI16 by Nutlin-3, a DNA damage agent, in HCC cells in vitro, and the potential mechanisms. Human HCC SMMC-7721 (wild-type TP53), Huh-7 (mutant TP53), Hep3B (null TP53) and normal fetal liver L02 cell lines were examined. DSB damage in HCC cells was detected via γH2AX expression and foci formation assay. The expression of IFI16 and IFNB mRNA was measured using RT-PCR, and subcellular localization and expression of the IFI16 protein were detected using chromatin fractionation, Western blot analysis, and fluorescence microscopy. Treatment of SMMC-7721 cells with Nutlin-3 (10 μmol/L) or etoposide (40 μmol/L) induced significant DSB damage. In SMMC-7721 cells, Nutlin-3 significantly increased the expression levels of IFI16 and IFNB mRNA, and partially redistributed chromatin-bound IFI16 protein to the cytoplasm. These effects were blocked by pretreatment with pifithrin-α, a p53 inhibitor. Furthermore, Nutlin-3 did not induce ectopic expression of IFI16 protein in Huh-7 and Hep3B cells. Moreover, the association of IFI16 with chromatin and Nutlin-3-induced changes in localization were not detected in L02 cells. Nutlin-3 regulates the subcellular localization of IFI16 in HCC cells in vitro in a p53-dependent manner.

Trisomy 21-Associated Abnormalities in IGF Signalling and the Fetal Microenvironment Both Contribute to Disruption of Fetal Hematopoiesis in Down Syndrome

In Down syndrome (DS), trisomy 21 (T21) causes perturbation of fetal liver (FL) hematopoiesis leading to expansion of megakaryocyte-erythroid (MK-E) progenitors. On this background, FL hematopoietic stem/progenitor cells (HSPC) may acquire GATA1 gene mutations which usually present as a neonatal preleukemic condition, Transient Myeloproliferative Disorder (TMD). TMD develops only in fetal life and most cases spontaneously regress after birth, suggesting unique features of the T21 fetal microenvironment may be important in driving both abnormal DS FL hematopoiesis and mutant GATA1 clones in TMD. Previous work showed activation of insulin-like growth factor (IGF) signalling in TMD and DS-AMKL and fetal-specific IGF dependence of murine MK development implicating T21 in altered fetal IGF signalling.To investigate the role of IGF signalling in fetal MK-E development in DS FL without GATA1 mutations, we performed gene expression profiling (GEP) of DS FL (n=5) and normal FL CD34+ cells (n=3). GSEA showed significant enrichment of IGF targets. We therefore measured expression of the principal IGF receptor, IGF1R, on DS FL HSPC (n=6). Surface IGF1R was uniformly expressed on all DS FL HSPC subpopulations. However, there was no difference in IGF1R surface or gene expression between DS and normal FL HSPC (n=8) suggesting that increased IGF1R expression on TMD and DS-AMKL cells is an early event and may reflect their fetal rather than T21 origin. To determine if the enrichment of IGF targets in DS FL HSPC might reflect increased IGF production in the FL microenvironment, we compared IGF expression in DS FL mesenchymal stromal cells (MSC)(n=4) and hepatocytes with normal FL MSC (n=4). Both DS and normal FL MSC expressed large amounts of IGF2, but little or no IGF1 with no difference between DS and normal MSC. Similarly, IGF2 was expressed by IHC at similar levels in both DS and normal fetal hepatocytes and MSC whereas IGF1 was barely detectable. Normal and DS fetal bone marrow (BM) MSC also expressed high levels of IGF2, but not IGF1 in contrast to adult BM MSC. Thus, IGF2 is the main IGF produced in the human fetal hematopoietic environment. Consistent with a specific role in DS FL hematopoiesis, IGF2 caused marked proliferation of DS FL clonogenic MK and MK-E cells (n=3) and stimulated MK proliferation in liquid culture while in normal FL, IGF2 stimulated EPO-independent BFU-E and Pre-BFU-E but had no effect on MK cells. Since differential effects of IGF2 on DS/normal FL HSPC might be due to altered expression of the Insulin Receptor (InsR), which binds IGF2 with low affinity, we compared InsR surface and gene expression in DS and normal FL HSPC but found no significant difference. IGF2 also binds to IGF2R, a negative regulator of IGF2, however there was no difference in IGF2R expression between DS and normal FL HSPC. Co-culture of normal FL CD34+ cells with DS or normal FL MSC (n=3) promoted MK and erythroid cell proliferation and differentiation. Since the effect of DS FL MSC on normal FL CD34+ cells was significantly greater (~4-fold) than that of normal FL MSC, the data above suggest this is unlikely to be due to IGF2 alone. To investigate whether T21 alters the MSC secretome, we performed GEP of DS FL MSC (n=3) and compared this with normal FL MSC (n=5). Both normal FL and DS FL expressed known hematopoietic cytokine genes, including SCF, CXCL12, G-CSF, M-CSF, Flt3 and IL-6, as well as IGF2 but not IGF1, EPO or TPO. Of the top 80 differentially expressed genes (Fold Change>2), ~40% encode secreted proteins, including several which affect IGF availability.Conclusion: T21 perturbs FL hematopoiesis through both HSPC-intrinsic mechanisms, including changes in IGF responsiveness, and through alterations to the FL microenvironment. DisclosuresNo relevant conflicts of interest to declare.

Downregulated protein O-fucosyl transferase 1 (Pofut1) expression exerts antiproliferative and antiadhesive effects on hepatocytes by inhibiting Notch signalling

The Notch signalling pathway is essential for proper cell growth and development. Many factors interact in the cellular context to bring about its effects. Critical to this process is the influence of Pofut1 whose function is to fucosylate Notch receptors and ligands on the cell surface for proper interaction. Of the three liver cell lines, HepG2, SMMC-7721 (hepatoma cell lines) and L-02 (normal fetal liver cell line) were investigated. In the current study, Pofut1 was silenced in L-02 due to its significantly high level of expression, (P<0.05) using transient SiRNA. Notch1, Cyclin D1 and p53 were significantly suppressed consequently. The effect on cell cycle, proliferation, adhesion and migration were investigated. Evidence adduced, indicate a general modification of cellular function. While proliferation and adhesion were significantly inhibited, the cell cycle arrest was obvious (P<0.05), migration was not affected. The effects seen are akin to those reported in previous studies in hepatoma cells mimicking some of the effects of notch1 silencing. Results of this study indicate a possible role of Pofut1 conferring a tumor suppressor role through the Notch signalling pathway.

Betaine homocysteine methyltransferase (BHMT)-dependent remethylation pathway in human healthy and tumoral liver

Carcinogenesis is a multi-step and multifactorial process. It includes genetic, epigenetic, nutritional and environmental factors, which are closely interconnected. Human hepatocellular carcinoma (HCC) is among the most frequent and lethal cancers. Imbalance in the S-adenosylmethionine (SAM) concentration, the main methyl group donor, strongly influences the development of HCC. Key enzymes of carbon metabolism are greatly reduced in patients with cirrhosis and HCC. These alterations play a role in genetic instability and epigenetic modifications (DNA methylation, and histone modifications), however, the molecular underlying mechanisms are still poorly understood. We aimed to investigate betaine homocysteine methyltransferase (BHMT) expression in HepG2 cells and human hepatocarcinoma tissues. Tumor and surrounding healthy tissue were compared. HepG2 cells and tumor samples showed a strong decrease in BHMT transcripts resulting from the transcription of a splicing variant that contained a frameshift mutation generating a premature termination codon and gene loss of function. This splicing variant, not detected in normal adult and fetal liver, cannot be explained by any mechanism involving the known splicing consensus sequences. BHMT activity was abolished in HepG2 cells and protein expression was detected neither in HepG2 cells nor in five of the six tumor samples investigated. Further investigation is needed to elucidate whether this abnormal BHMT transcription is part of cause or consequence of liver carcinogenesis.

Neonatal iron overload and tissue siderosis due to gestational alloimmune liver disease

Gestational alloimmune liver disease is the main cause of the neonatal hemochromatosis phenotype, wherein severe neonatal liver disease is associated with iron overload and extrahepatic tissue siderosis. How fetal liver disease produces extrahepatic siderosis is not known. We hypothesized that fetal liver injury causes deficient hepcidin production and poor regulation of placental iron flux. Under the resulting conditions of iron overload, the tissue pattern of extrahepatic siderosis is determined by the normal expression of proteins involved in the import of non-transferrin-bound iron and the export of cellular iron. Liver and extrahepatic tissues from infants with gestational alloimmune liver disease were examined and compared to normal age-appropriate tissues. Serum iron indices indicate iron overload and excess non-transferrin bound iron in gestational alloimmune liver disease. The diseased liver showed significantly reduced hepcidin, hemojuvulin, and transferrin gene expression compared to the normal fetal and neonatal liver. Those extrahepatic tissues that are typically involved in pathological siderosis in neonatal hemochromatosis, whether from normal or diseased newborns, consistently expressed solute carrier family 39 (zinc transporter), member 14 (ZIP14) for non-transferrin-bound iron uptake and expressed little ferroportin for iron export. Excess non-transferrin-bound iron in gestational alloimmune liver disease may result from fetal liver injury that causes reduced synthesis of key iron regulatory and transport proteins. Whereas, the pattern of extrahepatic siderosis appears to be determined by the normal capacity of various tissues to import non-transferrin-bound iron and not export cellular iron.

A splicing variant leads to complete loss of function of betaine–homocysteine methyltransferase (BHMT) gene in hepatocellular carcinoma

The remethylation of homocyteine into methionine is catalyzed either by methionine synthase (MTR) or by betaine–homocysteine methyltransferase (BHMT), in the liver. Choline/betaine deficiency and impaired BHMT pathway have been associated with hepatocellular carcinogenesis, in animal models. The molecular mechanisms that impair the BHMT pathway are unknown. We aimed to investigate BHMT, BHMT2, and MTR expression in HepG2 cells and human hepatocarcinoma tissues. Transcripts were quantified by RT-qPCR and splicing was assessed by analysis of exon junctions and sequencing of variants. Protein expression was studied by Western Blot, immunohistochemistry and enzyme activity. Tumor tissue was compared with surrounding healthy tissue. RT-qPCR of HepG2 cells and of tumor samples showed a strong decrease of transcripts of BHMT and BHMT2, compared to normal. MTR transcript levels were not different. The decreased BHMT expression resulted from the transcription of a splicing variant that produced a frameshift in exon 4, with a premature termination codon in exon 5 and a loss of function of the gene. This splicing variant did not fit with any mechanism resulting from known splicing consensus sequences and was not detected in normal adult and fetal liver. Consistently, BHMT activity was abolished in HepG2 and protein expression was not detectable in HepG2 and in 5 of the 6 tumor samples, compared to normal tissues. In conclusion, a transcription variant of exon 4 produces a loss of function of BHMT in human hepatocarcinoma. Whether this abnormal transcription of BHMT is part or consequence of liver carcinogenesis should deserve further investigations.

Development of Acute Megakaryoblastic Leukemia in Down Syndrome Is Associated with Sequential Epigenetic Changes That Target the Down Syndrome Critical Region on Chromosome 21,

Abstract 3451Down syndrome (DS) is associated with an increased frequency of Acute Megakaryoblastic Leukemias (AMKL). In the context of DS, AMKL is usually preceded by the development of a transient myeloproliferative disorder (TMD). Up to 10% of DS children may develop this disorder, and approximately 20–30% of those with TMD go on to develop AMKL, which may or may not be preceded by a return to normal hematopoiesis. While trisomy 21 (tri21) alone is not sufficient to develop AMKL, it is clear that the acquisition of somatic mutations in GATA1 is a requirement for the development of DS-AMKL. Much has been studied in relation with this genetic mutation, however not much is known about the epigenetic changes accompanying the development of AMKL in DS patients. Given the step-wise nature of the development of DS-AMKL, we hypothesized that distinct and cumulative epigenetic changes would be associated with each of the phases of the disease. In order to address this hypothesis we performed DNA methylation profiling of a cohort of 7 primary DS-AMKL samples using the latest design of the HELP microarray assay, which covers ∼230,000 CpG sites at ∼22,000 RefSeq promoter regions. In order to better understand how aberrant epigenetic patterns become established during the development of this disease we compared these DNA methylation profiles to those of mononuclear cells from normal fetal liver (FL-MNC) (n=4), tri21 FL-MNC (n=4) and TMD blasts (n=6). We identified a set of 711 unique genes that displayed significant loss of methylation in Tri21 FL-MNC with respect to control FL-MNC (FDR < 5% and methylation delta ≥ 25%). However, when tri21 FL-MNC were compared to the GATA1s positive TMD blasts, we observed that TMD samples acquired significant hypermethylation of 346 unique genes at the same significance cutoff. A direct comparison of the genes affected by these two opposing waves of methylation changes demonstrated that different sets of genes were being targeted at each stage. Next we compared the DNA methylation profiles of TMD and DS-AMKL blasts, and found them to be virtually identical, indicating that an epigenetic imprint becomes established at the TMD phase, which is still present at the leukemic phase. Finally we compared DS-AMKL to a cohort of 8 non-DS AMKL, and observed that DS-AMKL samples display significant hypomethylation of 267 unique genes, indicating that these two diseases are not only genetically but also epigenetically distinct. In determining the location of the epigenetic changes acquired at the different stages of the disease we found that, similar to what had been previously reported for gene expression, DNA methylation changes did not localize solely to chr 21, but that these changes were distributed along all chromosomes. However, detailed analysis of chr 21 revealed that changes on this chromosome preferentially targeted a specific region on the long arm, the Down Syndrome Critical Region (DSCR). This region becomes significantly hypomethylated in Tri21 FL-MNC and remains hypomethylated through all the stages of the disease. Several groups have sought to identify candidate genes on the DSCR that may contribute to the pathogenesis of the disease. Two genes, BACH1 and CXADR, have been identified in common by two different studies as being overexpressed in DS-AMKL vs. Non-DS AMKL. Both of these genes displayed aberrant hypomethylation at their promoter regions in Tri21 FL MNC, TMD and DS-AMKL samples: such hypomethylation was absent from all the control samples. Finally, given that GATA1 mutations are a requirement for the development of DS-AMKL, we compared the different aberrant DNA methylation signatures to the normal GATA1 targets identified by ChIP sequencing experiments, and found 20–25% overlap. In summary, we have completed the first comprehensive DNA methylation study of the developmental phases of DS-AMKL and have demonstrated that a) specific epigenetic changes are associated with the different stages of the disease, b) these changes occur in ‘waves’ of sequential hypo and hypermethylation, c) aberrant DNA methylation on chr 21 preferentially targets the DSCR and results in aberrant overexpression of the associated genes, and d) epigenetic profiles of TMD and DS-AMKL are virtually identical. These observations lead us to believe that an epigenetic memory established at the TMD stage may be at least in part contributing to the development of full-blown AMKL at a later time point. Disclosures:No relevant conflicts of interest to declare.