Human umbilical cord-derived mesenchymal stem cell (UC-MSC) transplantation has shown promise for the treatment of various diseases. For clinical applications, UC-MSCs have been stored in 0.9% saline, 5% dextrose, dextrose and sodium chloride injection, Plasma-Lyte A, 1% human serum albumin (1% HSA), or 5% HSA before administration, but the effect of storage conditions on the viability and biological function of the cells remains unknown. Freshly harvested UC-MSCs were resuspended and incubated in these solutions for 2, 4, or 6 h at 4°C or room temperature (24°C). Cell viability, apoptotic/necrotic fraction, poststorage growth potential, immunophenotype, immunosuppressive capacity, and differentiation capacity were analyzed. When stored in parenteral solutions, UC-MSCs showed progressive deterioration in survival viability and adhesion ability. After 6-h storage, the best viability and attachment rate of UC-MSCs decreased to 83.0 ± 1.6% and 71.8 ± 3.2%, respectively. Our results suggested that UC-MSCs in these conditions lose their viability in a short time. However, it seems that the other biological functions of the surviving UC-MSCs were little affected. Since UC-MSCs suspended in these mediums lose their survival viability in a short time to levels significantly below the permissible limits (70%) by FDA, precautions need to be taken on using these solutions as suspension medium and further studies on the optimal methods for preservation are urgent.

DNA methylation is thought to be involved in the etiology of neural tube defects (NTDs). However, the exact mechanism between DNA methylation and NTDs remains unclear. Herein, we investigated the change of methylation in mouse model of NTDs associated with folate dysmetabolism by use of ultraperformance liquid chromatography tandem mass spectrometry (UPLC/MS/MS), liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS), microarray, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and Real time quantitative PCR. Results showed that NTD neural tube tissues had lower concentrations of 5-methyltetrahydrofolate (5-MeTHF, P = 0.005), 5-formyltetrahydrofolate (5-FoTHF, P = 0.040), S-adenosylmethionine (SAM, P = 0.004) and higher concentrations of folic acid (P = 0.041), homocysteine (Hcy, P = 0.006) and S-adenosylhomocysteine (SAH, P = 0.045) compared to control. Methylation levels of genomic DNA decreased significantly in the embryonic neural tube tissue of NTD samples. 132 differentially methylated regions (35 low methylated regions and 97 high methylated regions) were selected by microarray. Two genes (Siah1b, Prkx) in Wnt signal pathway demonstrated lower methylated regions (peak) and higher expression in NTDs (P<0.05; P<0.05). Results suggest that DNA hypomethylation was one of the possible epigenetic variations correlated with the occurrence of NTDs induced by folate dysmetabolism and that Siah1b, Prkx in Wnt pathway may be candidate genes for NTDs.

Neural tube defects (NTDs) remain one of the most life-threatening birth defects affecting infants. Most patients with NTDs eventually develop lifelong disability, which cause significant morbidity and mortality and seriously reduce the quality of life. Our previous study has found that ethionine inhibits cell viability by disrupting the balance between proliferation and apoptosis, and preventing neural stem cells from differentiating into neurons and astrocytes. However, how ethionine participates in the pathogenesis of neural tube development through N6-methyladenosine (m6A) modification remains unknown. This study aims to investigate METTL3- and ALKBH5-mediated m6A modification function and mechanism in NTDs. Herein, our results demonstrate that SAM play not only a compensatory role, it also leads to changes of m6A modification in neural tube development and regulation. Additionally, these data implicate that METTL3 is enriched in HT-22 cells, and METTL3 knockdown reduces cell proliferation and increases apoptosis through suppressing Wnt/β-catenin signaling pathway. Significantly, overexpression of ALKBH5 can only inhibit cell proliferation, but cannot promote cell apoptosis. This research reveals an important role of SAM in development of NTDs, providing a good theoretical basis for further research on NTDs. This finding represents a novel epigenetic mechanism underlying that the m6A modification has profound and lasting implications for neural tube development.