Phytochemical evaluation, embryotoxic, and teratogenic effects of Buwakan (Decalobanthus peltatus (L.) A.R.Simões & Staples) leaf extracts on duck embryo.

Decalobanthus peltatus is a woody vine that is commonly utilized in traditional Southeast Asian medicinal preparations. Despite the documented therapeutic uses of D. peltatus, there is hardly any information regarding its toxic effects on its consumers. In this study, crude leaf extracts (aqueous, methanol, ethyl acetate, and hexane) from D. peltatus were prepared and evaluated for their embryotoxicity and teratogenic effects. Phytochemical screening of bioactive compounds from the plants showed the presence of alkaloids, flavonoids, saponins, steroids, and tannins. In addition, investigations on the toxicity of the crude leaf extracts were determined using brine shrimp lethality assay, in which the LC50 was calculated. Results showed that the ethyl acetate leaf extract was the most toxic among the crude leaf extracts, with an LC50 of 14.54 ppm. Based on this result, ethyl acetate leaf extract was treated on duck embryos, and the alteration of vascular branching patterns in the chorioallantoic membrane was quantified. Gross morphological and histological analysis of the skin tissues from the treated duck embryos were also examined. We found significant reduction of primary and tertiary vessel diameters in the duck embryos treated with ethyl acetate leaf extracts in both concentrations compared to the control group. Treated duck embryos exhibited gross malformations, growth retardation, and hemorrhages on the external body surfaces at 1000 ppm. Histopathological analysis of the skin tissues from the 14-day-old treated duck embryos showed a reduced number of feather follicles compared to the control group. These results suggest that D. peltatus crude leaf extracts present risks when taken in significant dosages and comprehensive toxicity testing on therapeutic herbs should be performed to ensure their safety on the consumers.

Reducing mitomycin-C-induced ROS levels in mouse feeder cells improves induced pluripotent stem cell colony growth.

Chemically defined stem cell culture media are often costly, and the use of mitotically arrested mouse embryonic fibroblasts (MEFs) as feeder cells is a popular and cost-efficient way to maintain induced pluripotent stem cells (iPSCs). However, the commonly used mitotic inhibitor mitomycin-C (MMC) is known to cause cellular metabolic stress. Therefore, our aim was to determine whether such stress in feeder cells indirectly affects iPSC growth during coculture. We report that prolonged exposure to MMC causes metabolic stress in MEFs in the form of oxidative dysregulation. Through optimization of MMC exposure time, we show how to effectively arrest MEFs without inducing oxidative stress, thus promoting significantly better colony growth rates (p < 0.05), improved viability and longer periods between passages of iPSCs in coculture.

CRISPR/Cas9-mediated gene correction in hemophilia B patient-derived iPSCs.

The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system is an efficient genome-editing tool that holds potential for gene therapy. Here, we report an application of this system for gene repair in hemophilia B (HB) using induced pluripotent stem cells (iPSCs). We prepared targeting plasmids with homology arms containing corrected sequences to repair an in-frame deletion in exon 2 of the factor IX (F9) gene and transfected patient-derived iPSCs with the Cas9 nuclease and a guide RNA expression vector. To validate the expression of corrected F9, we attempted to induce the differentiation of iPSCs toward hepatocyte-like cells (HLCs) in vitro. We successfully repaired a disease-causing mutation in HB in patient-derived iPSCs. The transcription product of corrected F9 was confirmed in HLCs differentiated from gene-corrected iPSCs. Although further research should be undertaken to obtain completely functional hepatocytes with secretion of coagulation factor IX, our study provides a proof-of-principle for HB gene therapy using the CRISPR/Cas9 system.

Functional analysis of ectodermal ß-catenin during external genitalia formation.

ß-catenin is a molecule belonging to the armadillo family of proteins that is a crucial core-component of cellular adherens junctions, and a component of the canonical Wnt-signaling pathway. We attempted to analyze the functional significance of ectodermal-derived ß-catenin during the development of the mouse genital tubercle, a mammalian anlage of the external genitalia. For this purpose, the conditional loss of function mouse mutant Wnt7a-Cre;ß-cat(f/f) was utilized. Loss of ectodermal ß-catenin leads to the formation of urethral cleft during preputial uprising. Although expression of E-cadherin was retained in the genital tubercle ectoderm of mutants, probably through plakoglobin compensatory expression, expression of other crucial adherens junction components such as α-catenin and F-actin in the cell-cell border were distinctly reduced. We also showed that ß-catenin is necessary for the expression of its transcriptional downstream target Lef-1 which was localized in the basal layer of the preputial ectoderm, excluding the midventral region at E15.5. Such specialized region was observed to possess cytoplasmic ß-catenin expression at this stage. Coincidentally, mitotically active cells were also found in the basal layer of the preputial ectoderm excluding the midventral region. In mutant genital tubercle, cell proliferation in the preputial ectoderm was decreased. Taken together, we suggest that ectodermal ß-catenin is necessary not only to maintain adherens junction integrity, but also to regulate cell proliferation possibly through Lef-1 functions. Thus, ß-catenin is shown to perform dual functions, initially as an adhesion molecule and later on as a possible transcription factor.