Spatial transcriptomics reveals gene interactions and signaling pathway dynamics in rat embryos with anorectal malformation.

Anorectal malformation (ARM) is a prevalent early pregnancy digestive tract anomaly. The intricate anatomy of the embryonic cloaca region makes it challenging for traditional high-throughput sequencing methods to capture location-specific information. Spatial transcriptomics was used to sequence libraries of frozen sections from embryonic rats at gestational days (GD) 14 to 16, covering both normal and ARM cases. Bioinformatics analyses and predictions were performed using methods such as WGCNA, GSEA, and PROGENy. Immunofluorescence staining was used to verify gene expression levels. Gene expression data was obtained with anatomical annotations of clusters, focusing on the cloaca region's location-specific traits. WGCNA revealed gene modules linked to normal and ARM cloacal anatomy development, with cooperation between modules on GD14 and GD15. Differential gene expression profiles and functional enrichment were presented. Notably, protein levels of Pcsk9, Hmgb2, and Sod1 were found to be downregulated in the GD15 ARM hindgut. The PROGENy algorithm predicted the activity and interplay of common signaling pathways in embryonic sections, highlighting their synergistic and complementary effects. A competing endogenous RNA (ceRNA) regulatory network was constructed from whole transcriptome data. Spatial transcriptomics provided location-specific cloaca region gene expression. Diverse bioinformatics analyses deepened our understanding of ARM's molecular interactions, guiding future research and providing insights into gene regulation in ARM development.

Mesenteric and omental lymphatic malformations in children: seven-year surgical experience from two centers in China.

PURPOSE: To compare the clinical characteristics, surgical management and prognosis of mesenteric lymphatic malformations (ML) and omental lymphatic malformations (OL) in children. METHODS: This retrospective study included 148 ML patients and 53 OL patients who underwent surgical treatment at two centers between January 2016 and December 2022. Details about the patients' clinical characteristics, cyst characteristics, preoperative complications, surgical methods, and prognosis were retrieved and compared. RESULTS: No significant differences in sex ratio, prenatal diagnosis, or age of diagnosis were noted between ML and OL patients. Vomiting was more common in ML patients than in OL patients (46.6% vs. 22.6%, P = 0.002), but OL patients were more likely to be misdiagnosed (35.8% vs. 18.9%, P = 0.012). The size of the cysts in OL patients was significantly larger than that in ML patients (14.0 [4.0-30.0] vs. 10.0 [2.0-50.0] cm, P<0.001), and cysts with turbid fluid were more common in OL patients (38.0% vs. 20.6%, P<0.001). More OL patients than ML patients had preoperative hemorrhage or infection of cysts (41.5% vs. 31.8%, P<0.016). Cyst excision was performed in 137 (92.6%) ML patients and 51 (96.2%) OL patients, and the incidence of postoperative complications was lower (12.6% vs. 4.2%, P = 0.165) among OL patients. The main postoperative complications included adhesive ileus and recurrence of cysts. Additionally, more OL patients than ML patients were treated with laparoscopic surgery (69.8% vs. 39.2%, P<0.001). CONCLUSIONS: There were differences in clinical characteristics, cyst characteristics and preoperative complications between ML and OL patients. Cyst excision was the most common surgical method that was used to treat both ML and OL patients, and laparoscopic surgery could be a feasible surgical approach for treating OL patients with a good prognosis. TRIAL REGISTRATION: Retrospectively registered.

Rack1-mediated ferroptosis affects hindgut development in rats with anorectal malformations: Spatial transcriptome insights.

Anorectal malformation (ARM), a common congenital anomaly of the digestive tract, is a result of insufficient elongation of the urorectal septum. The cytoplasmic protein Receptor of Activated C-Kinase 1 (Rack1) is involved in embryonic neural development; however, its role in embryonic digestive tract development and ARM formation is unexplored. Our study explored the hindgut development and cell death mechanisms in ARM-affected rats using spatial transcriptome analysis. We induced ARM in rats by administering ethylenethiourea via gavage on gestational day (GD) 10. On GDs 14-16, embryos from both normal and ARM groups underwent spatial transcriptome sequencing, which identified key genes and signalling pathways. Rack1 exhibited significant interactions among differentially expressed genes on GDs 15 and 16. Reduced Rack1 expression in the ARM-affected hindgut, verified by Rack1 silencing in intestinal epithelial cells, led to increased P38 phosphorylation and activation of the MAPK signalling pathway. The suppression of this pathway downregulated Nqo1 and Gpx4 expression, resulting in elevated intracellular levels of ferrous ions, reactive oxygen species (ROS) and lipid peroxides. Downregulation of Gpx4 expression in the ARM hindgut, coupled with Rack1 co-localisation and consistent mitochondrial morphology, indicated ferroptosis. In summary, Rack1, acting as a hub gene, modulates ferrous ions, lipid peroxides, and ROS via the P38-MAPK/Nqo1/Gpx4 axis. This modulation induces ferroptosis in intestinal epithelial cells, potentially influencing hindgut development during ARM onset.

Anti-miR-141-3p maintains homeostasis between autophagy and apoptosis by targeting Yy1 in the fetal lumbosacral defecation center of rats.

Anorectal malformations (ARMs) are congenital diseases that lead to postoperative fecal incontinence, constipation, and soiling, despite improvements in surgery; however, their pathological mechanisms remain unclear. Here, we report the role of microRNA-141-3p in maintaining homeostasis between apoptosis and autophagy in the lumbosacral defecation center of fetal rats with ARMs. Elevated microRNA-141-3p expression inhibited YIN-YANG-1 expression by binding its 3' UTR, and repressed autophagy and triggered apoptosis simultaneously. Then, adenylate cyclase 3 was screened to be the downstream target gene of YIN-YANG-1 by chromatin immunoprecipitation sequencing experiments, and Yin Yang 1 could positively activate the transcription of adenylate cyclase 3 by directly interacting with the motif GAGATGG and ATGG in its promoter. Intraamniotic microinjection of adeno-rno-microRNA-141-3p-sponge-GFP in fetal rats with ARMs on embryonic day 15 restored apoptosis-autophagy homeostasis. These findings reveal that microRNA-141-3p upregulation impaired homeostasis between apoptosis and autophagy by inhibiting the YIN-YANG-1/adenylate cyclase 3 axis, and that intraamniotic injection of anti-microRNA-141-3p helped maintain homeostasis in the lumbosacral defecation center of ARMs during embryogenesis.

Regulatory role of m6A epitranscriptomic modifications in normal development and congenital malformations during embryogenesis.

The discovery of N6-methyladenosine (m6A) methylation and its role in translation has led to the emergence of a new field of research. Despite accumulating evidence suggesting that m6A methylation is essential for the pathogenesis of cancers and aging diseases by influencing RNA stability, localization, transformation, and translation efficiency, its role in normal and abnormal embryonic development remains unclear. An increasing number of studies are addressing the development of the nervous and gonadal systems during embryonic development, but only few are assessing that of the immune, hematopoietic, urinary, and respiratory systems. Additionally, these studies are limited by the requirement for reliable embryonic animal models and the difficulty in collecting tissue samples of fetuses during development. Multiple studies on the function of m6A methylation have used suitable cell lines to mimic the complex biological processes of fetal development or the early postnatal phase; hence, the research is still in the primary stage. Herein, we discuss current advances in the extensive biological functions of m6A methylation in the development and maldevelopment of embryos/fetuses and conclude that m6A modification occurs extensively during fetal development. Aberrant expression of m6A regulators is probably correlated with single or multiple defects in organogenesis during the intrauterine life. This comprehensive review will enhance our understanding of the pivotal role of m6A modifications involved in fetal development and examine future research directions in embryogenesis.

A novel genotype-phenotype between persistent-cloaca-related VACTERL and mutations of 8p23 and 12q23.1.

The mechanism underlying anorectal malformations (ARMs)-related VACTERL (vertebral defects, anal atresia, cardiac defects, tracheo-esophageal fistula, and renal and limb abnormalities) remains unclear. Copy number variation (CNV) contributed to VACTERL pathogenicity. Here, we report a novel CNV in 8p23 and 12q23.1 identified in a case of ARMs-related VACTERL association. This 12-year-old girl presented a cloaca (urethra, vagina, and rectum opening together and sharing a single tube length), an isolated kidney, and a perpetuation of the left superior vena cava at birth. Her intelligence, growth, and development were slightly lower than those of normal children of the same age. Array comparative genomic hybridization revealed a 9.6-Mb deletion in 8p23.1-23.3 and a 0.52-Mb duplication in 12q23.1 in her genome. Furthermore, we reviewed the cases involving CNVs in patients with VACTERL, 8p23 deletion, and 12q23.1 duplication, and our case was the first displaying ARMs-related VACTERL association with CNV in 8p23 and 12q23.1. These findings enriched our understanding between VACTERL association and the mutations of 8p23 deletion and 12q23.1 duplication. IMPACT: This is a novel case of a Chinese girl with anorectal malformations (ARMs)-related VACTERL with an 8p23.1-23.3 deletion and 12q23.1 duplication. Cloaca malformation is presented with novel copy number variation in 8p23.1-23.3 deletion and 12q23.1 duplication.

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.

Diagnosis and management of fecal incontinence in children and adolescents.

Fecal incontinence (FI) is a commonly occurring disease of high concern. It is characterized by voluntary and involuntary defecation in children and adolescents. It is not only a physical disease but also a psychological and behavioral disorder. FI poses a serious burden on individuals and their families and therefore has become a social problem. Unfortunately, the management of FI among children is still a challenge because the etiology varies widely. Constipation has been found to be the most common cause, while sphincter dysfunction and neurogenic abnormalities may also play a role. Currently, no consensus guidelines exist, and the criteria for selecting optional methods remain unclear. It is therefore necessary to improve the efficacy of diagnosis and management strategies of FI in children. This review focused on the classification and etiology, discussed the diagnosis and management methods of FI in children and adolescents, and aimed to guide future studies.

Role and mechanisms of the NF-ĸB signaling pathway in various developmental processes.

Since the discovery of the nuclear factor kappa B (NF-ĸB) transcription factor 36 years ago, many studies have linked the NF-ĸB signaling pathway to pathological and physiological processes, such as inflammation, immune response, and tumorigenesis. However, as the NF-ĸB signaling pathway is evolutionarily conserved from flies to humans, an increasing number of studies have focused on the impact of NF-ĸB signaling on developmental processes. While our understanding of the mechanisms underlying NF-ĸB signaling involved in tissue and organ development is limited, the numerous studies conducted in recent years have provided preliminary insights into these molecular mechanisms. In this review, we summarize the latest information on the molecular mechanisms behind NF-ĸB signaling involved in tissue and organ development, highlighting the role and significance of the NF-ĸB signaling pathway in developmental processes. This review elucidates the fact that the development of nearly all tissues is associated with NF-ĸB signaling, either directly or indirectly.

Intra-amniotic transplantation of brain-derived neurotrophic factor-modified mesenchymal stem cells treatment for rat fetuses with spina bifida aperta.

BACKGROUND: Spina bifida aperta (SBA) is a relatively common clinical type of neural tube defect. Although prenatal fetal surgery has been proven to be an effective treatment for SBA, the recovery of neurological function remains unsatisfactory due to neuron deficiencies. Our previous results demonstrated that intra-amniotic transplanted bone marrow mesenchymal stem cells (BMSCs) could preserve neural function through lesion-specific engraftment and regeneration. To further optimize the role of BMSCs and improve the environment of defective spinal cords so as to make it more conducive to nerve repair, the intra-amniotic transplanted BMSCs were modified with brain-derived neurotrophic factor (BDNF-BMSCs), and the therapeutic potential of BDNF-BMSCs was verified in this study. METHODS: BMSCs were modified by adenovirus encoding a green fluorescent protein and brain-derived neurotrophic factor (Ad-GFP-BDNF) in vitro and then transplanted into the amniotic cavity of rat fetuses with spina bifida aperta which were induced by all-trans-retinoic acid on embryonic day 15. Immunofluorescence, western blot and real-time quantitative PCR were used to detect the expression of different neuron markers and apoptosis-related genes in the defective spinal cords. Lesion areas of the rat fetuses with spina bifida aperta were measured on embryonic day 20. The microenvironment changes after intra-amniotic BDNF-BMSCs transplantation were investigated by a protein array with 90 cytokines. RESULTS: We found that BDNF-BMSCs sustained the characteristic of directional migration, engrafted at the SBA lesion area, increased the expression of BDNF in the defective spinal cords, alleviated the apoptosis of spinal cord cells, differentiated into neurons and skin-like cells, reduced the area of skin lesions, and improved the amniotic fluid microenvironment. Moreover, the BDNF-modified BMSCs showed a better effect than pure BMSCs on the inhibition of apoptosis and promotion of neural differentiation. CONCLUSION: These findings collectively indicate that intra-amniotic transplanted BDNF-BMSCs have an advantage of promoting the recovery of defective neural tissue of SBA fetuses.

Serum exosomal coronin 1A and dynamin 2 as neural tube defect biomarkers.

No highly specific and sensitive biomarkers have been identified for early diagnosis of neural tube defects (NTDs). In this study, we used proteomics to identify novel proteins specific for NTDs. Our findings revealed three proteins showing differential expression during fetal development. In a rat model of NTDs, we used western blotting to quantify proteins in maternal serum exosomes on gestational days E18, E16, E14, and E12, in serum on E18 and E12, in neural tubes on E18 and E12, and in fetal neural exosomes on E18. The expression of coronin 1A and dynamin 2 was exosome-specific and associated with spina bifida aperta embryogenesis. Furthermore, coronin 1A and dynamin 2 were significantly downregulated in maternal serum exosomes (E12-E18), neural tubes, and fetal neural exosomes. Although downregulation was also observed in serum, the difference was not significant. Differentially expressed proteins were further analyzed in the serum exosomes of pregnant women during gestational weeks 12-40 using enzyme-linked immunosorbent assays. The findings revealed that coronin 1A and dynamin 2 showed potential diagnostic efficacy during gestational weeks 12-40, particularly during early gestation (12-18 weeks). Therefore, these two targets are used as candidate NTD screening and diagnostic biomarkers during early gestation. KEY MESSAGES: We used proteomics to identify novel proteins specific for NTDs. CORO1A and DNM2 showed exosome-specific expression and were associated with SBA. CORO1A and DNM2 were downregulated in maternal serum exosomes and FNEs. CORO1A and DNM2 showed good diagnostic efficacy for NTDs during early gestation. These two targets may have applications as NTD screening and diagnostic biomarkers.

Automated prediction of early spontaneous miscarriage based on the analyzing ultrasonographic gestational sac imaging by the convolutional neural network: a case-control and cohort study.

BACKGROUND: It is challenging to predict the outcome of the pregnancy when fetal heart activity is detected in early pregnancy. However, an accurate prediction is of importance for obstetricians as it helps to provide appropriate consultancy and determine the frequency of ultrasound examinations. The purpose of this study was to investigate the role of the convolutional neural network (CNN) in the prediction of spontaneous miscarriage risk through the analysis of early ultrasound gestational sac images. METHODS: A total of 2196 ultrasound images from 1098 women with early singleton pregnancies of gestational age between 6 and 8 weeks were used for training a CNN for the prediction of the miscarriage in the retrospective study. The patients who had positive fetal cardiac activity on their first ultrasound but then experienced a miscarriage were enrolled. The control group was randomly selected in the same database from the fetuses confirmed to be normal during follow-up. Diagnostic performance of the algorithm was validated and tested in two separate test sets of 136 patients with 272 images, respectively. Performance in prediction of the miscarriage was compared between the CNN and the manual measurement of ultrasound characteristics in the prospective study. RESULTS: The accuracy of the predictive model was 80.32% and 78.1% in the retrospective and prospective study, respectively. The area under the receiver operating characteristic curve (AUC) for classification was 0.857 (95% confidence interval [CI], 0.793-0.922) in the retrospective study and 0.885 (95%CI, 0.846-0.925) in the prospective study, respectively. Correspondingly, the predictive power of the CNN was higher compared with manual ultrasound characteristics, for which the AUCs of the crown-rump length combined with fetal heart rate was 0.687 (95%CI, 0.587-0.775). CONCLUSIONS: The CNN model showed high accuracy for predicting miscarriage through the analysis of early pregnancy ultrasound images and achieved better performance than that of manual measurement.

Transcriptome analysis reveals the spinal expression profiles of non-coding RNAs involved in anorectal malformations in rat fetuses.

BACKGROUND: Despite improvements in anorectal malformation (ARM) therapy, patients might still experience post-operative problems such as fecal incontinence, constipation, and soiling. In particular, the dysplasia of the lumbosacral spinal cord in ARM patients is a major disorder that affects fecal function post-operation. However, the pathological mechanisms involved are still unclear. METHODS: The non-coding RNAs (ncRNAs) in the lumbosacral spinal cord of fetal rats with ethylenethiourea-induced ARM were identified using RNA sequencing (RNA-seq) and examined to determine their potential function. The lumbosacral spinal cord was isolated on embryonic day 17 for subsequent RNA extraction and RNA-seq. The transcriptome data was analyzed using bioinformatics analysis, followed by validation using quantitative reverse transcription PCR. RESULTS: Compared to the control group, 26 differentially expressed microRNAs (DEMs; 22 upregulated, 4 downregulated) and 112 differentially expressed long non-coding RNAs (63 upregulated, 49 downregulated) were identified in the ARM group. Several DEMs related to development, namely miR-200a-3p, miR-200b-3p, miR-200c-3p, miR-200a-5p, and miR-429, were selected for further analysis. Notably, compared to the control, the relative expression of miR-200 family members was highly upregulated in ARM fetal rats. Furthermore, GO and KEGG enrichment and miRNA-transcription factor-lncRNA/mRNA network analysis was explored to show molecular mechanism underlying DEMs. CONCLUSIONS: Our findings suggest the involvement of ncRNAs, especially the miR-200 family members, in the pathogenesis of lumbosacral spinal cord dysplasia in ARM fetal rats.

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.

LIN28A polymorphisms and hepatoblastoma susceptibility in Chinese children.

Hepatoblastoma (HB) is the most prevalent primary hepatic cancer in children aged 6 months to 3 years. LIN28A is recurrently mutated in various diseases, and critically involved in tumorigenesis. However, a limited number of studies have examined the involvement of LIN28A polymorphisms in HB risk. We used the TaqMan assay to genotype four LIN28A polymorphisms (rs3811464 G>A, rs3811463 T>C, rs34787247 G>A, and rs11247957 G>A) in 275 Chinese children with HB and 1018 cancer-free controls from five medical centers in China. Their association with HB risk was evaluated on the basis of odds ratio (OR) and corresponding 95% confidence interval (CI). Overall, no significant associations were found in single locus and combine analysis. Interestingly, in the stratified analysis, we found that subjects with 1-3 risk genotypes were more likely to develop HB in patients ≥17 months of age (adjusted OR=1.76, 95% CI=1.04-2.98, P=0.034). The rs3811464 GA/AA genotypes were associated with decrease HB risk in patients with clinical stage III+IV disease (adjusted OR=0.50, 95% CI=0.26-0.96, P=0.038). Our results suggest that the LIN28A polymorphisms have a weak association with HB susceptibility in the Chinese children. LIN28A rs3811464 G>A may decrease HB risk in stage III+IV patients which need further validations with larger samples and different ethnicities.

Altered mRNA and lncRNA expression profiles in the striated muscle complex of anorectal malformation rats.

BACKGROUND: Striated muscle complex (SMC) dysplasia has been confirmed to contribute to postoperative defecation dysfunction of patients with anorectal malformations (ARMs). To date, the potential molecular mechanisms of SMC dysplasia underlying the development of ARMs have not been clearly explained. This study examined the expression profiles of mRNAs and lncRNAs in the malformed SMC of ARM rats using RNA sequencing (RNA-seq). METHODS: A rat model of ARMs was established by the intragastric administration of 1% ethylene thiourea (ETU) on an embryonic day 10 (E10). The rats were subjected to euthanasia and the SMC samples were collected on E19. The expression of mRNAs and lncRNAs was analyzed by RNA-seq on the Illumina HiSeq2500 platform. qRT-PCR was used to confirm the results of RNA-seq. RESULTS: Compared with the levels in control rats, 1408 mRNAs and 472 lncRNAs were differentially expressed in the SMC of E19 ARM rats. GO and KEGG pathway analyses showed that the top enriched GO terms were mainly related to muscle development and the enriched pathways were associated with muscle and synaptic development. Protein-protein interaction network analysis was also performed using the STRING database. The network map revealed the interaction between the WNT3 protein and NTRK1, NTF4, MUSK, and BMP5 proteins. Finally, the qRT-PCR results further confirmed the RNA-seq data. CONCLUSION: Our findings indicate the involvement of these dysregulated mRNAs and lncRNAs in the pathogenesis of SMC dysplasia in ARMs, providing a theoretical foundation for developing interventions to improve postoperative defecation function.

Sox10 Is a Specific Biomarker for Neural Crest Stem Cells in Immunohistochemical Staining in Wistar Rats.

OBJECTIVE: Neural crest stem cells (NCSCs) are prototypically migratory cells immigrating from the dorsal neural tube to specific embryonic sites where they generate a variety of cell types. A lot of biomarkers for NCSCs have been identified. However, which biomarkers are the most specific is still unclear. METHODS: The rat embryos harvested in embryonic day 9 (E9), E9.5, E10, E10.5, E11, E12, E13, and E14 were paraffin-embedded and sectioned in transverse. NCSCs were spatiotemporally demonstrated by immunohistochemical staining with RET, p75NTR, Pax7, and Sox10. NCSCs were isolated, cultured, and stained with RET, p75NTR, Pax7, and Sox10. RESULTS: In the paraffin sections of rat embryos, the immunohistochemical staining of RET, p75NTR, and Sox10 can all be used in demonstrating NCSCs. Sox10 was positive mainly in NCSCs while RET and p75NTR were positive not only in NCSCs but also in other tissue cells. In primary culture cells, Sox10 was mainly in the nucleus of NCSCs, RET was mainly in the membrane, and p75NTR was positive in cytoplasm and membrane. CONCLUSIONS: Sox10 is the specific marker for immunohistochemical staining of NCSCs in paraffin sections. In cultured cells, Sox10, p75NTR, and RET presented a similar staining effect.

Spatiotemporal expression of neurogenic locus notch homolog protein 1 in developing caudal spinal cord of fetuses with anorectal malformations from ETU-fed rats.

Complications, such as fecal soiling, incontinence, and constipation, are major health issues for patients with anorectal malformations (ARMs) after surgery. Dysplasia of the caudal spinal cord is an increasingly pivotal area in the field of postoperative complications for patients with ARMs. However, the existing research has not fully defined the mechanism underlying ARMs development. The neurogenic locus notch homolog (Notch) signaling pathway comprises several highly conserved proteins that are involved in spinal cord developmental processes. In the present study, the emerging role of Notch1 in fetal lumbosacral spinal cords was investigated in a rat model of ARMs using ethylene thiourea. Immunohistochemical staining, western blot and quantitative reverse transcription real-time polymerase chain reaction were utilized to analyze spatiotemporal expression of Notch1 on embryonic days (E) 16, E17, E19, and E21. The expression levels of the neuronal marker neurofilament and recombination signal-binding protein-J protein were evaluated for temporal correlations to Notch1 expression. The results implied that Notch1 expression was reduced in lumbosacral spinal cord neurons of ARMs embryos compared to control embryos. These results showed that, in ARMs embryos decreased Notch1 expression is related to the dysplasia of the caudal spinal cord during embryogenesis, indicating that Notch signaling may participate pathogenic embryonic lumbosacral spinal development and may be associated with postoperative complications of ARMs.

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.

Transamniotic mesenchymal stem cell therapy for neural tube defects preserves neural function through lesion-specific engraftment and regeneration.

Neural tube defects (NTDs) lead to prenatal mortality and lifelong morbidity. Currently, surgical closure of NTD lesions results in limited functional recovery. We previously suggested that nerve regeneration was critical for NTD therapy. Here, we report that transamniotic bone marrow-derived mesenchymal stem cell (BMSC) therapy for NTDs during early development may achieve beneficial functional recovery. In our ex vivo rat embryonic NTD model, BMSCs injected into the amniotic cavity spontaneously migrated into the defective neural tissue. Hepatocyte growth factor and its receptor c-MET were found to play critical roles in this NTD lesion-specific migration. Using the in vivo rat fetal NTD model, we further discovered that the engrafted BMSCs specifically differentiated into the cell types of the defective tissue, including skin and different types of neurons in situ. BMSC treatment triggered skin repair in fetuses, leading to a 29.9 ± 5.6% reduction in the skin lesion area. The electrophysiological functional recovery assay revealed a decreased latency and increased motor-evoked potential amplitude in the BMSC-treated fetuses. Based on these positive outcomes, ease of operation, and reduced trauma to the mother and fetus, we propose that transamniotic BMSC administration could be a new effective therapy for NTDs.