Intra-amniotic mesenchymal stem cell therapy improves the amniotic fluid microenvironment in rat spina bifida aperta fetuses.

OBJECTIVES: Spina bifida aperta (SBA) is one of the most common neural tube defects. Neural injury in SBA occurs in two stages involving failed neural tube closure and progressive degeneration through contact with the amniotic fluid. We previously suggested that intra-amniotic bone marrow-derived mesenchymal stem cell (BMSC) therapy for fetal rat SBA could achieve beneficial functional recovery through lesion-specific differentiation. The aim of this study is to examine whether the amniotic fluid microenvironment can be improved by intra-amniotic BMSC transplantation. METHODS: The intra-amniotic BMSC injection was performed using in vivo rat fetal SBA models. The various cytokine expressions in rat amniotic fluid were screened by protein microassays. Intervention experiments were used to study the function of differentially expressed cytokines. RESULTS: A total of 32 cytokines showed significant upregulated expression in the BMSC-injected amniotic fluid. We focused on Activin A, NGF, BDNF, CNTF, and CXCR4. Intervention experiments showed that the upregulated Activin A, NGF, BDNF, and CNTF could inhibit apoptosis and promote synaptic development in fetal spinal cords. Inhibiting the activity of these factors weakened the anti-apoptotic and pro-differentiation effects of transplanted BMSCs. Inhibition of CXCR4 activity reduced the engraftment rate of BMSCs in SBA fetuses. CONCLUSION: BMSC transplantation can improve the amniotic fluid environment, and this is beneficial for SBA repair.

Neural Stem Cell-Derived Exosomal Netrin1 Contributes to Neuron Differentiation of Mesenchymal Stem Cells in Therapy of Spinal Bifida Aperta.

Spinal bifida aperta (SBA) is a congenital malformation with a high incidence. Bone marrow mesenchymal stem cell (BMSC) transplantation has the potential to repair the structure of damaged tissues and restore their functions. This is an optional treatment that can be used as a supplement to surgery in the treatment of SBA. However, the application of BMSCs is limited, as the neuronal differentiation rate of BMSCs is not satisfactory when used in treating severe SBA. Thus, we aimed to assess the effect of neural stem cell (NSC)-derived exosomes on BMSC neuronal differentiation and observe the therapeutic effect in an ex vivo rat SBA embryo model. We found that NSC-derived exosomes increased the neuronal differentiation rate of BMSCs in vitro and in the SBA embryo model ex vivo. Proteomic analysis showed that NSC-derived exosomes were enriched in Netrin1, which positively regulated neuronal differentiation. Netrin1 increased the neuronal differentiation rate of BMSCs and NSCs and upregulated the expression of the neuronal markers, microtubule-associated protein (Map2), neurofilament, and ß3-tubulin. Bioinformatic analysis revealed that Netrin1 treatment increased the expression of the transcription factors Hand2 and Phox2b, related to neuronal differentiation. Furthermore, the Netrin1-induced NSC neuronal differentiation was significantly blocked by Phox2b knockdown. We suggest that NSC-derived exosomal Netrin1 induces neuronal differentiation via the Hand2/Phox2b axis by upregulating the expression of Hand2 and Phox2b. Therefore, NSC-derived exosomes are a critical inducer of BMSC neuronal differentiation and represent a potential treatment agent that can benefit BMSC treatment in SBA.

Nuclear factor I-C disrupts cellular homeostasis between autophagy and apoptosis via miR-200b-Ambra1 in neural tube defects.

Impaired autophagy and excessive apoptosis disrupt cellular homeostasis and contribute to neural tube defects (NTDs), which are a group of fatal and disabling birth defects caused by the failure of neural tube closure during early embryonic development. However, the regulatory mechanisms underlying NTDs and outcomes remain elusive. Here, we report the role of the transcription factor nuclear factor I-C (NFIC) in maintaining cellular homeostasis in NTDs. We demonstrated that abnormally elevated levels of NFIC in a mouse model of NTDs can interact with the miR-200b promoter, leading to the activation of the transcription of miR-200b, which plays a critical role in NTD formation, as reported in our previous study. Furthermore, miR-200b represses autophagy and triggers apoptosis by directly targeting the autophagy-related gene Ambra1 (Autophagy/Beclin1 regulator 1). Notably, miR-200b inhibitors mitigate the unexpected effects of NFIC on autophagy and apoptosis. Collectively, these results indicate that the NFIC-miR-200b-Ambra1 axis, which integrates transcription- and epigenome-regulated miRNAs and an autophagy regulator, disrupts cellular homeostasis during the closure of the neural tube, and may provide new insight into NTD pathogenesis.

miR-351-3p promotes rat amniotic fluid-derived mesenchymal stromal cell proliferation via targeting the coding sequence of Abca4.

Amniotic fluid-derived mesenchymal stromal cells (AFMSCs) present different features, depending on the isolation timing and culture conditions. The lack of uniform experimental standards hinders the comparison of results from different studies on AFMSCs. Moreover, understanding the molecular mechanisms that underlie the features of AFMSCs isolated at different embryonic developmental stages might allow the obtention of more viable and highly proliferative AFMSCs through genetic modification. We isolated AFMSCs from pregnant rats at embryonic day (E)12, E15, E18, and E21 and compared their cell proliferation capacity and transcriptome. The cell counting kit-8 assay and RNA sequencing revealed that E12 and E15 AFMSCs showed different characteristics from E18 and E21 AFMSCs. Therefore, AFMSCs were divided into two groups: early (E12 and E15) and late (E18 and E21) pregnancy-stage groups. Next, we screened the gene/microRNA pair Abca4/miR-351-3p that was related to cell proliferation. Abca4 knockdown/overexpression suggested that this gene represses the proliferation of AFMSCs, which is a newly discovered function of this gene. Finally, dual luciferase reporter gene assays confirmed that miR-351-3p targeted the coding sequence of Abca4 and regulated AFMSC proliferation. miR-351-3p promotes AFMSC proliferation via targeting the coding sequence of Abca4. Our findings provide a molecular foundation for further research for obtaining AFMSCs with a higher proliferation capacity.

Complement factors and alpha-fetoprotein as biomarkers for noninvasive prenatal diagnosis of neural tube defects.

Neural tube defects (NTDs) are serious congenital malformations. In this study, we aimed to identify more specific and sensitive maternal serum biomarkers for noninvasive NTD screenings. We collected serum from 37 pregnant women carrying fetuses with NTDs and 38 pregnant women carrying normal fetuses. Isobaric tags for relative and absolute quantitation were conducted for differential proteomic analysis, and an enzyme-linked immunosorbent assay was used to validate the results. We then used a support vector machine (SVM) classifier to establish a disease prediction model for NTD diagnosis. We identified 113 differentially expressed proteins; of these, 23 were either up- or downregulated 1.5-fold or more, including five complement proteins (C1QA, C1S, C1R, C9, and C3); C3 and C9 were downregulated significantly in NTD groups. The accuracy rate of the SVM model of the complement factors (including C1QA, C1S, and C3) was 62.5%, with 60% sensitivity and 67% specificity, while the accuracy rate of the SVM model of alpha-fetoprotein (AFP, an established biomarker for NTDs) was 62.5%, with 75% sensitivity and 50% specificity. Combination of the complement factor and AFP data resulted in the SVM model accuracy of 75%, and receiver operating characteristic curve analysis showed 75% sensitivity and 75% specificity. These data suggest that a disease prediction model based on combined complement factor and AFP data could serve as a more accurate method of noninvasive prenatal NTD diagnosis.

miR-322 treatment rescues cell apoptosis and neural tube defect formation through silencing NADPH oxidase 4.

AIMS: Failure of neural tube closure resulting from excessive apoptosis leads to neural tube defects (NTDs). NADPH oxidase 4 (NOX4) is a critical mediator of cell growth and death, yet its role in NTDs has never been characterized. NOX4 is a potential target of miR-322, and we have previously demonstrated that miR-322 was involved in high glucose-induced NTDs. In this study, we investigated the effect of NOX4 on the embryonic neuroepithelium in NTDs and reveal a new regulatory mechanism for miR-322 that disrupts neurulation by ameliorating cell apoptosis. METHODS: All-trans-retinoic acid (ATRA)-induced mouse model was utilized to study NTDs. RNA pull-down and dual-luciferase reporter assays were used to confirm the interaction between NOX4 and miR-322. In mouse neural stem cells and whole-embryo culture, Western blot and TUNEL were carried out to investigate the effects of miR-322 and NOX4 on neuroepithelium apoptosis in NTD formation. RESULTS: NOX4, as a novel target of miR-322, was upregulated in ATRA-induced mouse model of NTDs. In mouse neural stem cells, the expression of NOX4 was inhibited by miR-322; still further, NOX4-triggered apoptosis was also suppressed by miR-322. Moreover, in whole-embryo culture, injection of the miR-322 mimic into the amniotic cavity attenuated cell apoptosis in NTD formation by silencing NOX4. CONCLUSION: miR-322/NOX4 plays a crucial role in apoptosis-induced NTD formation, which may provide a new understanding of the mechanism of embryonic NTDs and a basis for potential therapeutic target against NTDs.

Expression profile of maternal circulating microRNAs as non-invasive biomarkers for prenatal diagnosis of congenital heart defects.

OBJECTIVE: The discovery of cell free fetal microRNAs (miRNAs) in maternal circulation has opened up novel probabilities for non-invasive prenatal diagnosis. This study aims to investigate circulating miRNAs as potential biomarkers in the serum of pregnant women with congenital heart defect (CHD) fetuses. METHOD: A total of 110 pregnant women including 50 CHD cases and 60 healthy controls were included in this study. miRNA microarray followed by real-time PCR was used to explore miRNA expression. Receiver operating characteristic (ROC) curves were calculated to assess the diagnostic capability of miRNAs for fetal CHDs. RESULTS: 38 Serum miRNAs were revealed to be differentially expressed in the CHD group as compared to control group via microarray. Among these, nine down-regulated and three up-regulated miRNAs were validated by real-time PCR. Ten of these miRNAs were rapidly reduced in the normal maternal serum after delivery as compared to before delivery. In particular, we identified a biomarker panel consisting of four miRNAs (miR-142-5p, miR-1275, miR-4666a-3p and miR-3664-3p) capable of distinguishing CHDs from controls (area under the ROC curve (AUC), 0.920; p < 0.0001). CONCLUSION: The discovery of these dysregulated pregnancy-associated miRNAs in maternal serum may be potential biomarkers for non-invasive prenatal diagnosis of fetal CHDs.

Comprehensive maternal serum proteomics identifies the cytoskeletal proteins as non-invasive biomarkers in prenatal diagnosis of congenital heart defects.

Congenital heart defects (CHDs) are the most common group of major birth defects. Presently there are no clinically used biomarkers for prenatally detecting CHDs. Here, we performed a comprehensive maternal serum proteomics assessment, combined with immunoassays, for the discovery of non-invasive biomarkers for prenatal diagnosis of CHDs. A total of 370 women were included in this study. An isobaric tagging for relative and absolute quantification (iTRAQ) proteomic approach was used first to compare protein profiles in pooled serum collected from women who had CHD-possessing or normal fetuses, and 47 proteins displayed significant differential expressions. Targeted verifications were performed on 11 proteins using multiple reaction monitoring mass spectrometry (MRM-MS), and the resultant candidate biomarkers were then further validated using ELISA analysis. Finally, we identified a biomarker panel composed of 4 cytoskeletal proteins capable of differentiating CHD-pregnancies from normal ones [with an area under the receiver operating characteristic curve (AUC) of 0.938, P < 0.0001]. The discovery of cytoskeletal protein changes in maternal serum not only could help us in prenatal diagnosis of CHDs, but also may shed new light on CHD embryogenesis studies.

Identification of PCSK9 as a novel serum biomarker for the prenatal diagnosis of neural tube defects using iTRAQ quantitative proteomics.

To identify candidate serum molecule biomarkers for the non-invasive early prenatal diagnosis of neural tube defects (NTDs), we employed an iTRAQ-based quantitative proteomic approach to analyze the proteomic changes in serum samples from embryonic day (E) 11 and E13 pregnant rats with spina bifida aperta (SBA) induced by all-trans retinoic acid. Among the 390 proteins identified, 40 proteins at E11 and 26 proteins at E13 displayed significant differential expression in the SBA groups. We confirmed 5 candidate proteins by ELISA. We observed the space-time expression changes of proprotein convertase subtilisin/kexin type 9 (PCSK9) at different stages of fetal development, including a marked decrease in the sera of NTD pregnancies and gradual increase in the sera of normal pregnancies with embryonic development. PCSK9 demonstrated the diagnostic efficacy of potential NTD biomarkers [with an area under the receiver operating characteristic curve of 0.763, 95% CI: 065-0.88]. Additionally, PCSK9 expression in the spinal cords and placentas of SBA rat fetuses was markedly decreased. PCSK9 could serve as a novel molecular biomarker for the non-invasive prenatal screening of NTDs and may be involved in the pathogenesis of NTDs at critical periods of fetal development.

Sensory neuron differentiation potential of in utero mesenchymal stem cell transplantation in rat fetuses with spina bifida aperta.

BACKGROUND: In previous studies, we found that the deficiency of sensory and motor neurons was a primary defect associated with the spinal malformation. Upon prenatal treatment of spina bifida through in utero stem cell transplantation in a retinoic acid-induced spina bifida rat model, we found that the mesenchymal stem cell (MSCs) survived, migrated, and differentiated into cells of a neural lineage. In the present study, we investigated whether the transplanted MSCs had the potential to differentiate into sensory neurons or to protect sensory neurons in the defective spinal cord. METHODS: Pregnant rats treated with retinoic acid on embryonic day (E) 10, underwent fetal surgery for MSC transplantation on E16. The fetuses were harvested on E20. Immunofluorescence was used to detect the expression of Brn3a protein in the transplanted MSCs and dorsal root ganglion (DRG) neurons in the defective spinal cords. The expression of the transcription factors Brn3a and Runx1 in spinal cords was analyzed using real-time polymerase chain reaction. RESULTS: Some of the transplanted MSCs expressed sensory neuron cell specific phenotypes. The expression of Brn3a and Runx1 was upregulated in the defective spinal cords when compared to controls. The percentage of Brn3a-positive neurons in DRG was also increased after transplantation. CONCLUSION: Our results indicate that the transplantation of MSCs into the spinal cord could promote the transplanted MSCs and the surrounding cells to differentiate toward a sensory neuron cell fate and to play an important role in protecting sensory neurons in DRG. This approach might be of value in the treatment of sensory neuron deficiency in spina bifida aperta.

The association of the MTHFR c.1625A>C genetic variant with the risk of congenital heart diseases in the Chinese.

The purpose of this study is to investigate the association of methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms with the risk of congenital heart diseases (CHD). The genotypes of the MTHFR genetic variant were determined by the polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing methods. Our data suggested that the allelic and genotypic frequencies of CHD patients were significantly different from non-CHD controls. The MTHFR c.1625A>C genetic variant was significantly associated with the increased risk of CHD (CC vs. AA: odds ratio [OR]=2.29, 95% confidence interval [CI] 1.15-4.53, p=0.016; C vs. A: OR=1.47, 95% CI 1.11-1.96, p=0.008). Results from this study indicate that the MTHFR c.1625A>C genetic variant influences the risk of CHD in the studied population.

Up-regulated FHL1 expression maybe involved in the prognosis of Hirschsprung's disease.

BACKGROUND: In a subset of patients with Hirschsprung's disease (HSCR), gastrointestinal motor dysfunction persisted long after surgical correction. Gastrointestinal motility is achieved through the coordinated activity of the enteric nervous system, interstitial cells of Cajal, and smooth muscle (SMC) cells. Inhibition of four-and-a-half LIM protein-1 (Fhl1) expression by siRNA significantly decreases pulmonary artery SMCs migration and proliferation. Furthermore when up-expressing FHL1 in atrial myocytes, K (+) current density markedly increases, therefore changing myocytes' response to an electrical stimulus. However whether FHL1 in colon SMCs (the final effector organ) influences intestinal motility in HSCR patients has not been clarified. METHODS: FHL1 mRNA and protein expressions were analyzed in 32 HSCR colons and 4 normal colons. RESULTS: Smooth muscle layers were thicken and disorganized in HSCR. FHL1 was expressed in the ganglion cells of the myenteric plexus, submucosa, as well as in the longitudinal and circular muscle layer of the ganglionic colon. FHL1 mRNA relative expression level in aganglionic colons was 1.06 ± 0.49 (ganglionic colon relative expression level was 1) (P=0.44). FHL1 protein gray level relative to GAPDH in normal colons was 0.83 ± 0.09. FHL1 expression level in ganglionic colon (1.66 ± 0.30) or aganglionic colon (1.81 ± 0.35) was significantly higher than that in normal colons (P=0.045 and P=0.041, respectively). Meanwhile, we found FHL1 expression in aganglionic colon was slightly stronger than that in ganglionic colon (P=0.036). CONCLUSION: These data suggested that up-regulated FHL1 in smooth muscle in HSCR might be associated with intestinal wall remodeling in HSCR and might be one of the risk factors for gastrointestinal motor dysfunction.

Comparative proteomics of Hirschsprung's disease.

Hirschsprung's disease (HSCR) is a developmental disorder of the enteric nervous system characterized by aganglionosis in distal gut. The estimated population incidence of HSCR is 1/5000 live births, but higher in Asian populations. As the disease mainly manifested bowel motility disturbance, the underlying mechanism is unknown. Furthermore, in the long term up to 75% of patients showed unsatisfactory postoperative bowel function like incontinence or constipation, and 10% required a permanent colostomy. Improved therapy of HSCR is required, but the pathophysiological mechanism for postoperative bowel dysfunction is not clear. In this study, we perform a proteomics study in HSCR patients, expecting some findings in protein alterations to provide more information to reveal the pathophysiological mechanisms of disturbed bowel function before and after surgery therapy. As a result, we identified 16 proteins expressed differently in aganglionic segment of HSCR patients. These proteins function diversely, and included cytoskeleton proteins, regulatory proteins and some enzymes. BIOLOGICAL SIGNIFICANCE: In the present study, we performed a 2-DE based proteomic research on HSCR patients, in order to find some clue for the pathomechanism of bowel motility of HSCR disease. As a character of this study, we also compared the expression of altered proteins in ganglionic segment of HSCR patients with that in normal children. Our results showed that some altered proteins found in aganglionic segment had also changed their expression in ganglionic segment comparing with normal children. This result suggested that the ganglionic segment of HSCR patients was not completely normal, and this is important because it provided more information to understand the pathophysiological mechanisms of bowel dysfunction and will help to the therapy of HSCR disease.

Diagnostic role of microRNA expression profile in the serum of pregnant women with fetuses with neural tube defects.

The discovery of placental microRNAs (miRNAs) in maternal serum has opened up new possibilities for non-invasive prenatal diagnosis. However, the expression of miRNAs in the serum of pregnant women with fetuses with neural tube defects (NTDs) has not been characterized. In this article, we explored serum miRNAs as potential biomarkers in the serum of pregnant women with NTD fetuses. By using a miRNA microarray that covers 887 human miRNAs, we revealed 17 miRNAs with significant change in expression in serum of pregnant women with NTD fetuses and women with normal pregnancies. Quantitative reverse-transcription PCR (qRT-PCR) analysis validated that the expression for six miRNAs (miR-142-3p, miR-144, miR-720, miR-575, miR-765, and miR-1182) was up-regulated and that for miR-1275 was down-regulated. To determine whether these miRNAs were related to pregnancy, we compared the miRNA levels in pre- and post-delivery maternal serum samples. Six of these miRNAs were rapidly reduced in post-delivery serum (p < 0.05). Moreover, by receiver operating characteristic (ROC) curve analysis, the area under the ROC curve (AUC) of combining these six miRNAs was 0.803 (p < 0.001). Thus, we reveal six pregnancy-associated miRNAs that are deregulated in the serum of pregnant women with NTD fetuses and highlight the clinical potential of serum miRNAs as biomarkers for diagnosis and prognostication of fetal NTDs.

SEMA3A rs7804122 polymorphism is associated with Hirschsprung disease in the Northeastern region of China.

BACKGROUND: Hirschsprung disease (HSCR) is a congenital disorder characterized by an absence of intrinsic ganglion cells in the nerve plexuses of the lower colon. Our previous results showed increased semaphorin 3A (SEMA3A) expression may be the risk factor for HSCR pathology in a subset of patients. Therefore, the association between polymorphisms in SEMA3A and the risk of HSCR was examined. METHODS: The genotypes of two SNPs (rs7804122 and rs797821) in the SEMA3A gene in 119 patients with HSCR and 93 controls were examined using PCR-sequencing to determine the contribution of SEMA3A to the HSCR phenotype. PCR reaction with cDNA template was also used to find out whether a novel mutation (Chr7:83634610A→T) influences the SEMA3A pre-mRNA splicing. RESULTS: Genotypes comprising allele G of rs7804122 (GG or AG) were over-represented in patients (48.74 vs. 24.8%; p = 0.0013) which indicated that the risk of HSCR was significantly higher among subjects with the GG or AG genotype than among the subjects with the AA genotype. No statistically significant associations were found for SNP rs797821 at the allele or genotype levels. The differences in genotypes and allele distributions of rs7804122 and rs797821 between various clinical classifications were not statistically significant. The novel heterozygous mutation (Chr7:83634610A→T) 30bp away from an intron/exon boundary, had no detectable effect on splicing efficiency. CONCLUSION: Our results for rs7804122 provided preliminary evidence that the SEMA3A gene is involved in the susceptibility to HSCR in the Northeastern Chinese population.

Homeobox C9 is not potentially related to congenital heart disease in Chinese patients.

BACKGROUND: Congenital heart disease (CHD) is one of the most common human birth defects. The etiology and pathogenesis of CHD are complex and involve several genes as well as multiple changes in signaling pathways. The aim of this study was to identify potential pathological mutations in the Homeobox C9 (Hoxc9) gene in 350 Chinese children with CHD to further understand the etiology of CHD. METHOD: Sequence analysis of the Hoxc9 gene in 350 nonsyndromic patients with CHD Result: We did not identify any nonsynonymous variants in the coding regions of Hoxc9 in the patients with CHD. We found one synonymous variant c.C564T (p. his188his) in one ventricular septal defect patient. We also identified four previously reported polymorphisms (rs56368105, rs12817092, rs34079606, and rs2241820) in CHD. CONCLUSIONS: We did not find any diagnostic alterations in the coding regions of Hoxc9 among the patients with CHD. Nevertheless, to our knowledge, this is the first study of Hoxc9 in nonsyndromic CHD and has expanded our overall knowledge of the etiology of this disease.

Semaphorin 3A expression in the colon of Hirschsprung disease.

BACKGROUND: Hirschsprung disease (HSCR) is a congenital disorder characterized by an absence of intrinsic ganglion cells in the nerve plexuses of the lower colon. The Semaphorin 3A (SEMA3A) gene is involved in the migration of enteric neural precursors (ENPs). To analyze the function of SEMA3A in HSCR, the SEMA3A expression in different colon segments in HSCR was examined. METHODS: The expression levels of SEMA3A in both ganglionic and aganglionic colon tissues of 32 patients with HSCR and in colon tissue of 5 newborn unaffected individuals were examined by real-time RT-PCR, Western-blot, and immunohistology. RESULTS: Comparison of SEMA3A expression levels between ganglionic and aganglionic tissues in HSCR revealed upregulation of SEMA3A expression in 43.75% (14/32) of the aganglionic colons. SEMA3A was expressed in the ganglion cells of the myenteric plexus, submucosa, as well as in the longitudinal and circular muscle layer of the normal colon of both unaffected newborns and patients with HSCR. In the aganglionic segment of patients with HSCR, SEMA3A was highly expressed in the circular muscle layer and was also detected in the submucosa and in the longitudinal muscles layer. The fluorescence intensity of SEMA3A in the circular muscle layer in the aganglionic segment was much higher than that in ganglionic segment (p < .001). CONCLUSION: SEMA3A expression was upregulated in the aganglionic smooth muscle layer of the colon in some patients with HSCR and our data suggest that increased SEMA3A expression may be a risk factor for HSCR pathology in a subset of patients.