Changes in the osmolarity of the embryonic microenvironment induce neural tube defects.

Many maternal disorders that modify the embryonic microenvironment, such as a change in osmolarity, can affect development, but how these changes influence the early embryo remains obscure. Neural tube defects, for example, are common congenital disorders found in fetus and neonates. In this study, we investigated the impact of anisotonic osmolarity (unequal osmotic pressures) on neural tube development in the early chick embryo, finding that neuronal cell differentiation was impaired in the neural tube due to enhanced apoptosis and repressed cell proliferation. Anisotonic osmolarity also affected normal development of the neural crest, which in turn influenced abnormal development of the neural tube. As neural tube development is highly dependent on the proper expression of bone morphogenetic protein 4 (BMP4), paired box 7 (PAX7), and sonic hedgehog (SHH) genes in the dorsal and ventral regions along the tube, we investigated the impact of anisotonic osmolarity on their expression. Indeed, small changes in osmolarity could positively and negatively impact the expression of these regulatory genes, which profoundly affected neural tube development. Thus, both the central and peripheral nervous systems were perturbed by anisotonic consitions as a consequence of the abnormal expression of key genes within the developing neural tube.

microRNA-1 inhibits cardiomyocyte proliferation in mouse neonatal hearts by repressing CCND1 expression.

BACKGROUND: The functions of microRNA-1 (miR-1) in cardiac hypertrophy, and cardiomyocyte differentiation have been investigated. However, the mechanism on how miR-1 could repress cardiomyocyte proliferation has not been fully elucidated. METHODS: We address this issue by investigating whether miR-1 affected the proliferation of neonatal cardiomyocyte and identify some of the genes targeted by miR-1. miR-1 was over-expressed in neonatal cardiomyocytes and the effect on cell cycle and growth were analyzed by flow cytometry and Brdu-incorporation assay. Relevant vectors carrying the luciferase reporter were constructed for validation of miR-1 binding to its matching sites on the 3'-untranslated region of the predicated target mRNAs. Cardiomyocytes were co-transfected with the vectors and miR-1 mimics, then luciferase reporter assay was performed. Lastly, we examined the expression of target genes in cardiomyocytes after transfection with miR-1 mimics, as well as their normal expression pattern in 2- and 13-day-old mice hearts. RESULTS: We have demonstrated that miR-1 was the most significantly upregulated miRNA in 13-day-old mouse hearts compared with 2-day-old hearts. We also showed that miR-1 could repress cardiomyocyte G1/S phase transition, proliferation and viability. IGF1 and CCND1 were identified as candidate target genes regulated by miR-1. In addition, overexpression of miR-1 could suppress the expression of these two genes at the mRNA level. It could also correspondingly inhibit CCND1 expression at the protein level but not for IGF1. CONCLUSIONS: Our results suggest that miR-1 plays an important role in inhibiting cardiomyocyte proliferation in the developing neonatal mouse heart by directly suppressing the cell-cycle regulator, CCND1.

Transient acid treatment cannot induce neonatal somatic cells to become pluripotent stem cells.

Currently, there are genetic- and chemical-based methods for producing pluripotent stem cells from somatic cells, but all of them are extremely inefficient.  However, a simple and efficient technique has recently been reported by Obokata et al (2014a, b) that creates pluripotent stem cells through acid-based treatment of somatic cells.  These cells were named stimulus-triggered acquisition of pluripotency (STAP) stem cells. This would be a major game changer in regenerative medicine if the results could be independently replicated. Hence, we isolated CD45 (+) splenocytes from five-day-old Oct4-GFP mice and treated the cells with acidified (pH 5.7) Hank's Balanced Salt Solution (HBSS) for 25 min, using the methods described by Obokata et al 2014c. However, we found that this method did not induce the splenocytes to express the stem cell marker Oct4-GFP when observed under a confocal microscope three to six days after acid treatment. qPCR analysis also confirmed that acid treatment did not induce the splenocytes to express the stemness markers Oct4, Sox2 and Nanog.  In addition, we obtained similar results from acid-treated Oct4-GFP lung fibroblasts. In summary, we have not been able to produce STAP stem cells from neonatal splenocytes or lung fibroblasts using the acid-based treatment reported by Obokata et al (2014a, b, c).