Transcriptomic analysis reveals the anti-cancer effect of gestational mesenchymal stem cell secretome.

The environment created during embryogenesis contributes to reducing aberrations that drive structural malformations and tumorigenesis. In this study, we investigate the anti-cancer effect of mesenchymal stem cells (MSCs) derived from 2 different gestational tissues, the amniotic fluid (AF) and the chorionic villi (CV), with emphasis on their secretome. Transcriptomic analysis was performed on patient-derived AF- and CV-MSCs collected during prenatal diagnosis and identified both mRNAs and lncRNAs, involved in tissue homeostasis and inhibiting biological processes associated with the etiology of aggressive cancers while regulating immune pathways shown to be important in chronic disorders. Secretome enrichment analysis also identified soluble moieties involved in target cell regulation, tissue homeostasis, and cancer cell inhibition through the highlighted Wnt, TNF, and TGF-ß signaling pathways. Transcriptomic data were experimentally confirmed through in vitro assays, by evaluating the anti-cancer effect of the media conditioned by AF- and CV-MSCs and the exosomes derived from them on ovarian cancer cells, revealing inhibitory effects in 2D (by reducing cell viability and inducing apoptosis) and in 3D conditions (by negatively interfering with spheroid formation). These data provide molecular insights into the potential role of gestational tissues-derived MSCs as source of anti-cancer factors, paving the way for the development of therapeutics to create a pro-regenerative environment for tissue restoration following injury, disease, or against degenerative disorders.

Disruption of Fuz in mouse embryos generates hypoplastic hindbrain development and reduced cranial nerve ganglia.

BACKGROUND: The brain and spinal cord formation is initiated in the earliest stages of mammalian pregnancy in a highly organized process known as neurulation. Environmental or genetic interferences can impair neurulation, resulting in clinically significant birth defects known collectively as neural tube defects. The Fuz gene encodes a subunit of the CPLANE complex, a macromolecular planar polarity effector required for ciliogenesis. Ablation of Fuz in mouse embryos results in exencephaly and spina bifida, including dysmorphic craniofacial structures due to defective cilia formation and impaired Sonic Hedgehog signaling. RESULTS: We demonstrate that knocking Fuz out during embryonic mouse development results in a hypoplastic hindbrain phenotype, displaying abnormal rhombomeres with reduced length and width. This phenotype is associated with persistent reduction of ventral neuroepithelial stiffness in a notochord adjacent area at the level of the rhombomere 5. The formation of cranial and paravertebral ganglia is also impaired in these embryos. CONCLUSIONS: This study reveals that hypoplastic hindbrain development, identified by abnormal rhombomere morphology and persistent loss of ventral neuroepithelial stiffness, precedes exencephaly in Fuz ablated murine mutants, indicating that the gene Fuz has a critical function sustaining normal neural tube development and neuronal differentiation.

Neural tube defects and epigenetics: role of histone post-translational histone modifications.

Neural tube defects (NTDs) are the most common congenital anomalies of the CNS. It is widely appreciated that both genetic and environmental factors contribute to their etiology. The inability to ascribe clear genetic patterns of inheritance to various NTD phenotypes suggests it is possible that epigenetic mechanisms are involved in the etiology of NTDs. In this context, the contribution of DNA methylation as an underlying contributing factor to the etiology of NTDs has been extensively reviewed. Here, an updated accounting of the evidence linking post-translational histone modifications to these birth defects, relying heavily upon studies in humans, and the possible molecular implications inferred from reports based on cellular and animal models, are presented.

De novo heterozygous missense variants in CELSR1 as cause of fetal pleural effusions and progressive fetal hydrops.

Fetal hydrops as detected by prenatal ultrasound usually carries a poor prognosis depending on the underlying aetiology. We describe the prenatal and postnatal clinical course of two unrelated female probands in whom de novo heterozygous missense variants in the planar cell polarity gene CELSR1 were detected using exome sequencing. Using several in vitro assays, we show that the CELSR1 p.(Cys1318Tyr) variant disrupted the subcellular localisation, affected cell-cell junction, impaired planar cell polarity signalling and lowered proliferation rate. These observations suggest that deleterious rare CELSR1 variants could be a possible cause of fetal hydrops.

The novel linkage between Fuz and Gpr161 genes regulates sonic hedgehog signaling during mouse embryonic development.

Sonic hedgehog (Shh) signaling regulates embryonic morphogenesis utilizing primary cilia, the cell antenna acting as a signaling hub. Fuz, an effector of planar cell polarity (PCP) signaling, involves Shh signaling via cilia formation, while the G protein-coupled receptor 161 (Gpr161) is a negative regulator of Shh signaling. The range of phenotypic malformations observed in mice bearing mutations in either of these two genes is similar; however, their functional relations have not been previously explored. This study identified the genetic and biochemical link between Fuz and Gpr161 in mouse embryonic development. Fuz was genetically epistatic to Gpr161 via Shh signaling during mouse embryonic development. The FUZ biochemically interacted with GPR161, and Fuz regulated Gpr161 ciliary trafficking via ß-arrestin2. Our study suggested the novel Gpr161-Fuz axis that regulates Shh signaling during mouse embryonic development.

Dolutegravir-induced neural tube defects in mice are folate responsive.

OBJECTIVES: In 2018, the Botswana Tsepamo Study reported a nine-fold increased risk of neural tube defects in infants whose mothers were treated with dolutegravir (DTG) from the time of conception. As maternal folate supplementation and status is a well known modifier of neural tube defect (NTD) risk, we sought to evaluate birth outcomes in mice fed normal and low folic acid diets treated with DTG during pregnancy. DESIGN: DTG was evaluated for developmental toxicity using pregnant mice fed normal or low folic acid diet. METHODS: CD-1 mice were provided diet with normal (3 mg/kg) or low (0.3 mg/kg) folic acid. They were treated with water, a human therapeutic-equivalent dose, or supratherapeutic dose of DTG from mouse embryonic day E6.5 to E12.5. Pregnant dams were sacrificed at term (E18.5) and fetuses were inspected for gross, internal, and skeletal defects. RESULTS: Fetuses with exencephaly, an NTD, were present in both therapeutic human equivalent and supratherapeutic exposures in dams fed low folic acid diet. Cleft palates were also found under both folate conditions. CONCLUSIONS: Recommended dietary folic acid levels during mouse pregnancy ameliorate developmental defects that arise from DTG exposure. Since low folate status in mice exposed to DTG increases the risk for NTDs, it is possible that DTG exposures in people living with HIV with low folate status during pregnancy may explain, at least in part, the elevated NTD risk signal observed in Botswana. Based on these results, future studies should consider folate status as a modifier for DTG-associated NTD risk.

Folate regulation of planar cell polarity pathway and F-actin through folate receptor alpha.

Folate deficiency contribute to neural tube defects (NTDs) which could be rescued by folate supplementation. However, the underlying mechanisms are still not fully understood. Besides, there is considerable controversy concerning the forms of folate used for supplementation. To address this controversy, we prepared culture medium with different forms of folate, folic acid (FA), and 5-methyltetrahydrofolate (5mTHF), at concentrations of 5 µM, 500 nM, 50 nM, and folate free, respectively. Mouse embryonic fibroblasts (MEFs) were treated with different folates continuously for three passages, and cell proliferation and F-actin were monitored. We determined that compared to 5mTHF, FA showed stronger effects on promoting cell proliferation and F-actin formation. We also found that FOLR1 protein level was positively regulated by folate concentration and the non-canonical Wnt/planar cell polarity (PCP) pathway signaling was significantly enriched among different folate conditions in RNA-sequencing analyses. We demonstrated for the first time that FOLR1 could promote the transcription of Vangl2, one of PCP core genes. The transcription of Vangl2 was down-regulated under folate-deficient condition, which resulted in a decrease in PCP activity and F-actin formation. In summary, we identified a distinct advantage of FA in cell proliferation and F-actin formation over 5mTHF, as well as demonstrating that FOLR1 could promote transcription of Vangl2 and provide a new mechanism by which folate deficiency can contribute to the etiology of NTDs.

Pax3 lineage-specific deletion of Gpr161 is associated with spinal neural tube and craniofacial malformations during embryonic development.

Sonic hedgehog (Shh) signaling is the morphogen signaling that regulates embryonic craniofacial and neural tube development. G protein-coupled receptor 161 (Gpr161) is a negative regulator of Shh signaling, and its inactivation in mice results in embryo lethality associated with craniofacial defects and neural tube defects. However, the structural defects of later embryonic stages and cell lineages underlying abnormalities have not been well characterized due to the limited lifespan of Gpr161 null mice. We found that embryos with Pax3 lineage-specific deletion of Gpr161 presented with tectal hypertrophy (anterior dorsal neuroepithelium), cranial vault and facial bone hypoplasia (cranial neural crest), vertebral abnormalities (somite) and the closed form of spina bifida (posterior dorsal neuroepithelium). In particular, the closed form of spina bifida was partly due to reduced Pax3 and Cdx4 gene expression in the posterior dorsal neural tubes of Gpr161 mutant embryos with decreased Wnt signaling, whereas Shh signaling was increased. We describe a previously unreported role for Gpr161 in the development of posterior neural tubes and confirm its role in cranial neural crest- and somite-derived skeletogenesis and midbrain morphogenesis in mice.

A mutation in TBXT causes congenital vertebral malformations in humans and mice.

T-box transcription factor T (TBXT; T) is required for mesodermal formation and axial skeletal development. Although it has been extensively studied in various model organisms, human congenital vertebral malformations (CVMs) involving T are not well established. Here, we report a family with 15 CVM patients distributed across four generations. All affected individuals carry a heterozygous mutation, T c.596A>G (p.Q199R), which is not found in unaffected family members, indicating co-segregation of the genotype and phenotype. In vitro assays show that T p.Q199R increases the nucleocytoplasmic ratio and enhances its DNA-binding affinity, but reduces its transcriptional activity compared to the wild-type. To determine the pathogenicity of this mutation in vivo, we generated a Q199R knock-in mouse model that recapitulates the human CVM phenotype. The heterozygous Q199R mice show subtle kinked or shortened tails, while the homozygous mice exhibit tail filaments and severe vertebral deformities. Overall, we show that the Q199R mutation in T causes CVM in humans and mice, providing new evidence supporting the function of T in the genetic etiology of human CVM.

Transcriptomic analysis of stem cells from chorionic villi uncovers the impact of chromosomes 2, 6 and 22 in the clinical manifestations of Down syndrome.

BACKGROUND: Down syndrome (DS) clinical multisystem condition is generally considered the result of a genetic imbalance generated by the extra copy of chromosome 21. Recent discoveries, however, demonstrate that the molecular mechanisms activated in DS compared to euploid individuals are more complex than previously thought. Here, we utilize mesenchymal stem cells from chorionic villi (CV) to uncover the role of comprehensive functional genomics-based understanding of DS complexity. METHODS: Next-generation sequencing coupled with bioinformatic analysis was performed on CV obtained from women carrying fetuses with DS (DS-CV) to reveal specific genome-wide transcriptional changes compared to their euploid counterparts. Functional assays were carried out to confirm the biological processes identified as enriched in DS-CV compared to CV (i.e., cell cycle, proliferation features, immunosuppression and ROS production). RESULTS: Genes located on chromosomes other than the canonical 21 (Ch. 2, 6 and 22) are responsible for the impairment of life-essential pathways, including cell cycle regulation, innate immune response and reaction to external stimuli were found to be differentially expressed in DS-CV. Experimental validation confirmed the key role of the biological pathways regulated by those genes in the etiology of such a multisystem condition. CONCLUSIONS: NGS dataset generated in this study highlights the compromised functionality in the proliferative rate and in the innate response of DS-associated clinical conditions and identifies DS-CV as suitable tools for the development of specifically tailored, personalized intervention modalities.

Pax3 lineage-specific deletion of Gpr161 is associated with spinal neural tube and craniofacial malformations during embryonic development.

Shh signaling is the morphogen signaling that regulates embryonic craniofacial and neural tube development. G protein-coupled receptor 161 (Gpr161) is a negative regulator of Shh signaling, and its inactivation in mice results in embryo lethality with craniofacial and neural tube defects (NTDs). However, the structural defects of later embryonic stages in Gpr161 null mice and cell lineages underlying abnormalities were not well characterized due to their limited lifespan. We found the Pax3 lineage-specific deletion of Gpr161 in mice presented with tectal hypertrophy (anterior dorsal neuroepithelium), cranial vault and facial bone hypoplasia (cranial neural crest (CNC)), vertebral abnormalities (somite), and the closed form of spina bifida (posterior dorsal neuroepithelium). In particular, the closed form of spina bifida is partly due to the reduced Pax3 and Cdx4 gene expression of the posterior dorsal neural tubes of Gpr161 mutant embryos involving decreased Wnt signaling whereas Shh signaling was increased. This study provides the novel role of Gpr161 in the posterior neural tube development and confirms its role on CNC- and somite-derived skeletogenesis and midbrain morphogenesis in mice.

Maternal metabolism influences neural tube closure.

Changes in maternal nutrient availability due to diet or disease significantly increase the risk of neural tube defects (NTDs). Because the incidence of metabolic disease continues to rise, it is urgent that we better understand how altered maternal nutrient levels can influence embryonic neural tube development. Furthermore, primary neurulation occurs before placental function during a period of histiotrophic nutrient exchange. In this review we detail how maternal metabolites are transported by the yolk sac to the developing embryo. We discuss recent advances in understanding how altered maternal levels of essential nutrients disrupt development of the neuroepithelium, and identify points of intersection between metabolic pathways that are crucial for NTD prevention.

A non-coding insertional mutation of Grhl2 causes gene over-expression and multiple structural anomalies including cleft palate, spina bifida and encephalocele.

Orofacial clefts, including cleft lip and palate (CL/P) and neural tube defects (NTDs) are among the most common congenital anomalies, but knowledge of the genetic basis of these conditions remains incomplete. The extent to which genetic risk factors are shared between CL/P, NTDs and related anomalies is also unclear. While identification of causative genes has largely focused on coding and loss of function mutations, it is hypothesized that regulatory mutations account for a portion of the unidentified heritability. We found that excess expression of Grainyhead-like 2 (Grhl2) causes not only spinal NTDs in Axial defects (Axd) mice but also multiple additional defects affecting the cranial region. These include orofacial clefts comprising midline cleft lip and palate and abnormalities of the craniofacial bones and frontal and/or basal encephalocele, in which brain tissue herniates through the cranium or into the nasal cavity. To investigate the causative mutation in the Grhl2Axd strain, whole genome sequencing identified an approximately 4 kb LTR retrotransposon insertion that disrupts the non-coding regulatory region, lying approximately 300 base pairs upstream of the 5' UTR. This insertion also lies within a predicted long non-coding RNA, oriented on the reverse strand, which like Grhl2 is over-expressed in Axd (Grhl2Axd) homozygous mutant embryos. Initial analysis of the GRHL2 upstream region in individuals with NTDs or cleft palate revealed rare or novel variants in a small number of cases. We hypothesize that mutations affecting the regulation of GRHL2 may contribute to craniofacial anomalies and NTDs in humans.

Whole-exome sequencing study of hypospadias.

Hypospadias results from the impaired urethral development, which is influenced by androgens, but its genetic etiology is still unknown. Through whole exome sequencing analysis, we identified NR5A1, SRD5A2, and AR as mutational hotspots in the etiology of severe hypospadias, as these genes are related to androgen signaling. Additionally, rare damaging variants in cilia-related outer dynein arm heavy chain (ODNAH) genes (DNAH5, DNAH8, DNAH9, DNAH11, and DNAH17) (p = 8.5 × 10-47) were significantly enriched in hypospadias cases. The Dnah8 KO mice exhibited significantly decreased testosterone levels, which had an impact on urethral development and disrupted steroid biosynthesis. Combined with trios data, transcriptomic, and phenotypical and proteomic characterization of a mouse model, our work links ciliary genes with hypospadias. Overall, a panel of ODNAH genes with rare damaging variants was identified in 24% of hypospadias patients, providing significant insights into the underlying pathogenesis of hypospadias as well as genetic counseling.

Dual functions of TET1 in germ layer lineage bifurcation distinguished by genomic context and dependence on 5-methylcytosine oxidation.

Gastrulation begins when the epiblast forms the primitive streak or becomes definitive ectoderm. During this lineage bifurcation, the DNA dioxygenase TET1 has bipartite functions in transcriptional activation and repression, but the mechanisms remain unclear. By converting mouse embryonic stem cells (ESCs) into neuroprogenitors, we defined how Tet1-/- cells switch from neuroectoderm fate to form mesoderm and endoderm. We identified the Wnt repressor Tcf7l1 as a TET1 target that suppresses Wnt/ß-catenin and Nodal signalling. ESCs expressing catalytic dead TET1 retain neural potential but activate Nodal and subsequently Wnt/ß-catenin pathways to generate also mesoderm and endoderm. At CpG-poor distal enhancers, TET1 maintains accessible chromatin at neuroectodermal loci independently of DNA demethylation. At CpG-rich promoters, DNA demethylation by TET1 affects the expression of bivalent genes. In ESCs, a non-catalytic TET1 cooperation with Polycomb represses primitive streak genes; post-lineage priming, the interaction becomes antagonistic at neuronal genes, when TET1's catalytic activity is further involved by repressing Wnt signalling. The convergence of repressive DNA and histone methylation does not inhibit neural induction in Tet1-deficient cells, but some DNA hypermethylated loci persist at genes with brain-specific functions. Our results reveal versatile switching of non-catalytic and catalytic TET1 activities based on genomic context, lineage and developmental stage.

Neural tube defects: Prevalence, mortality, and maternal characteristics in two departmental hospitals in the northwestern region of Nicaragua, 2006-2018.

BACKGROUND: Congenital anomalies are the fifth most common cause of neonatal mortality in Nicaragua, and neural tube defects (NTDs) are the most common of all cases of lethality associated with a birth defect. Prevalence and mortality estimates are needed to propose effective intervention strategies that prevent NTDs over time. METHODS: A cross-sectional study was carried out in northwestern Nicaragua from January 2006 to December 2018. All cases of NTDs (anencephaly, spina bifida, and encephalocele) were registered in hospital surveillance systems, and the medical histories of the mothers and newborns were reviewed. Prevalence was calculated by considering the number of live births and stillbirths older than 20 weeks of gestation with NTDs, divided by the total number of live births and stillbirths in each study year. Neonatal mortality rate (NMR) for NTD, and case fatality for spina bifida was calculated. RESULTS: Two hundred fifty cases of NTDs were identified from 178,498 deliveries (177,316 live births and 1,182 stillbirths). The prevalence of NTDs during this time period was 14.01 (95% CI: 12.27-15.74) per 10,000 births. The prevalence of spina bifida (n = 140), anencephaly (n = 97), and encephalocele (n = 13) was 7.84, (95% CI: 6.54-9.14), 5.43 (95% CI: 4.30-6.45), and 0.73 (95% CI: 0.33-1.12) per 10,000 births, respectively. Mothers with fetus or newborns affected with NTDs did not use folic acid prior to conception, and 11% experienced periods of hyperthermia during the first trimester of pregnancy. NMR for NTDs was 0.55 per 1.000 livebirths. Case fatality for all NTDs and for spina bifida were 55% and 18%, respectively. CONCLUSION: The prevalence and mortality of NTDs in the northwestern region of Nicaragua present peaks and troughs during the study period. Spina bifida was the most frequent type of NTD. We believe that these findings could be of use by health policy makers to strengthen the primary prevention of NTDs in the region through the monitoring of the food fortification policy and folic acid supplementation to women of childbearing age. Additional etiologic studies of NTDs should be considered to identify additional prevention measures.

Exome-wide assessment of isolated biliary atresia: A report from the National Birth Defects Prevention Study using child-parent trios and a case-control design to identify novel rare variants.

The etiology of biliary atresia (BA) is unknown, but recent studies suggest a role for rare protein-altering variants (PAVs). Exome sequencing data from the National Birth Defects Prevention Study on 54 child-parent trios, one child-mother duo, and 1513 parents of children with other birth defects were analyzed. Most (91%) cases were isolated BA. We performed (1) a trio-based analysis to identify rare de novo, homozygous, and compound heterozygous PAVs and (2) a case-control analysis using a sequence kernel-based association test to identify genes enriched with rare PAVs. While we replicated previous findings on PKD1L1, our results do not suggest that recurrent de novo PAVs play important roles in BA susceptibility. In fact, our finding in NOTCH2, a disease gene associated with Alagille syndrome, highlights the difficulty in BA diagnosis. Notably, IFRD2 has been implicated in other gastrointestinal conditions and warrants additional study. Overall, our findings strengthen the hypothesis that the etiology of BA is complex.

Impaired Function of Solute Carrier Family 19 Leads to Low Folate Levels and Lipid Droplet Accumulation in Hepatocytes.

Low serum folate levels are inversely related to metabolic associated fatty liver disease (MAFLD). The role of the folate transporter gene (SLC19A1) was assessed to clarify its involvement in lipid accumulation during the onset of MAFLD in humans and in liver cells by genomic, transcriptomic, and metabolomic techniques. Genotypes of 3 SNPs in a case-control cohort were initially correlated to clinical and serum MAFLD markers. Subsequently, the expression of 84 key genes in response to the loss of SLC19A1 was evaluated with the aid of an RT2 profiler-array. After shRNA-silencing of SLC19A1 in THLE2 cells, folate and lipid levels were measured by ELISA and staining techniques, respectively. In addition, up to 482 amino acids and lipid metabolites were semi-quantified in SLC19A1-knockdown (KD) cells through ultra-high-performance liquid chromatography coupled with mass spectrometry. SNPs, rs1051266 and rs3788200, were significantly associated with the development of fatty liver for the single-marker allelic test. The minor alleles of these SNPs were associated with a 0.6/-1.67-fold decreased risk of developing MAFLD. When SLC19A1 was KD in THLE2 cells, intracellular folate content was four times lower than in wild-type cells. The lack of functional SLC19A1 provoked significant changes in the regulation of genes associated with lipid droplet accumulation within the cell and the onset of NAFLD. Metabolomic analyses showed a highly altered profile, where most of the species that accumulated in SLC19A1-KD-cells belong to the chemical groups of triacylglycerols, diacylglycerols, polyunsaturated fatty acids, and long chain, highly unsaturated cholesterol esters. In conclusion, the lack of SLC19A1 gene expression in hepatocytes affects the regulation of key genes for normal liver function, reduces intracellular folate levels, and impairs lipid metabolism, which entails lipid droplet accumulation in hepatocytes.