Supercapacitance/Resistance Behaviors of Helminth Eggs as Reliable Recognition and Direct Differentiation Probe

Parasitic helminths are usually known as undesired pathogens, causing various diseases in both human and animal species. In this study, we explore supercapacitance/resistance behaviors as a novel probe for rapid identification and direct differentiation of Fasciola hepatica, Parascaris equorum (with and without larvae), Dicrocoelium dendriticum, Taenia multiceps, and Moniezia expansa eggs. This claim is attributed to some characteristics, such as grave supercapacitance/area, high-energy storage/area, large power/egg, huge permittivity, and great electrical break-down potential, respectively (Fasciola hepatica: 2,158, 0.485, 2.7 × 10–3, 267, 52.6, Parascaris equorum without larvae: 2,825, 0.574, 3.0 × 10–3, 351, 68.4, Parascaris equorum with larvae: 4,519, 0.716, 2.4 × 10–3, 1.96, 97.6, Dicrocoelium dendriticum: 1,581, 0.219, 2.8 × 10–3, 1.96, 48.8, Moniezia expansa: 714, 0.149, 2.2 × 10–3, 0.88, 35.2, Taenia multiceps: 3,738, 0.619, 4.7 × 10–3, 4.63, 84.4), and durable capacitance up to at least 15,000 sequential cycles at different scan rates (between 2.0 × 10−4 and 120.0 V s−1) as well as highly differentiated resistance between 400 and 600 Ω. These traits are measured by the “Blind Patch-Clamp” method, at the giga ohm sealed condition (6.18 ± 0.12 GΩ cm−1, n = 5). Significant detection ranges are detected for each capacitance and resistance with gradient limits as large as at least 880 to 1,000 mF and 400 to 600 Ω depending on the type of helminth egg. The effect of water in the structure of helminth eggs has also been investigated with acceptable reproducibility (RSD 7%–10%, n = 5). These intrinsic characteristics would provide novel facilitators for direct helminth egg identification in comparison with several methods, such as ELISA, PCR, and microscopic methods.

Rapid Production and Genetic Stability of Human Mesenchymal Progenitor Cells Derived from Human Somatic Cell Nuclear Transfer-Derived Pluripotent Stem Cells

Pluripotent stem cell-derived mesenchymal progenitor cells (PSC-MPCs) are primarily derived through two main methods: three-dimensional (3D) embryoid body-platform (EB formation) and the 2D direct differentiation method. We recently established somatic cell nuclear transfer (SCNT)-PSC lines and showed their stemness. In the present study, we produced SCNT-PSC-MPCs using a novel direct differentiation method, and the characteristics, gene expression, and genetic stability of these MPCs were compared with those derived through EB formation. The recovery and purification of SCNT-PSC-Direct-MPCs were significantly accelerated compared to those of the SCNT-PSC-EB-MPCs, but both types of MPCs expressed typical surface markers and exhibited similar proliferation and differentiation potentials. Additionally, the analysis of gene expression patterns using microarrays showed very similar patterns. Moreover, array CGH analysis showed that both SCNT-PSC-Direct-MPCs and SCNT-PSC-EB-MPCs exhibited no significant differences in copy number variation (CNV) or single-nucleotide polymorphism (SNP) frequency. These results indicate that SCNT-PSC-Direct-MPCs exhibited high genetic stability even after rapid differentiation into MPCs, and the rate at which directly derived MPCs reached a sufficient number was higher than that of MPCs derived through the EB method. Therefore, we suggest that the direct method of differentiating MPCs from SCNT-PSCs can improve the efficacy of SCNT-PSCs applied to allogeneic transplantation.

Rapid target validation in a Cas9-inducible hiPSC derived kidney model

Recent advances in induced pluripotent stem cells (iPSCs), genome editing technologies and 3D organoid model systems highlight opportunities to develop new in vitro human disease models to serve drug discovery programs. An ideal disease model would accurately recapitulate the relevant disease phenotype and provide a scalable platform for drug and genetic screening studies. Kidney organoids offer a high cellular complexity that may provide greater insights than conventional single-cell type cell culture models. However, genetic manipulation of the kidney organoids requires prior generation of genetically modified clonal lines, which is a time and labor consuming procedure. Here, we present a methodology for direct differentiation of the CRISPR-targeted cell pools, using a doxycycline-inducible Cas9 expressing hiPSC line for high efficiency editing to eliminate the laborious clonal line generation steps. We demonstrate the versatile use of genetically engineered kidney organoids by targeting the autosomal dominant polycystic kidney disease (ADPKD) genes: PKD1 and PKD2. Direct differentiation of the respective knockout pool populations into kidney organoids resulted in the formation of cyst-like structures in the tubular compartment. Our findings demonstrated that we can achieve > 80% editing efficiency in the iPSC pool population which resulted in a reliable 3D organoid model of ADPKD. The described methodology may provide a platform for rapid target validation in the context of disease modeling.

Airway stem cells sense hypoxia and differentiate into protective solitary neuroendocrine cells

Neuroendocrine (NE) cells are epithelial cells that possess many of the characteristics of neurons, including the presence of secretory vesicles and the ability to sense environmental stimuli. The normal physiologic functions of solitary airway NE cells remain a mystery. We show that mouse and human airway basal stem cells sense hypoxia. Hypoxia triggers the direct differentiation of these stem cells into solitary NE cells. Ablation of these solitary NE cells during hypoxia results in increased epithelial injury, whereas the administration of the NE cell peptide CGRP rescues this excess damage. Thus, we identify stem cells that directly sense hypoxia and respond by differentiating into solitary NE cells that secrete a protective peptide that mitigates hypoxic injury.

A new alternative for corneal endothelial regeneration using autologous dental pulp stem cells

Background Failure of the corneal endothelium cell (CEC) monolayer is the main cause leading to corneal transplantation. Autologous cell-based therapies are required to reconstruct in vitro the cell monolayer. For this purpose, we propose the use of dental pulp stem cells isolated from the third molars to form CEC monolayer. We hypothesize that by using dental pulp stem cells (DPSC) that share an embryological origin with CEC, as they both arise from the neural crest, may allow a direct differentiation process avoiding the use of reprogramming techniques, such as induced pluripotent stem cells (iPSC). Methods In this work, we report a two-step differentiation protocol, where dental pulp stem cells are derived into neural crest stem cells and, then, into CEC. Results Initially, we compared the efficiency of direct differentiation of DPSC with the differentiation of iPSC to express NCSC related genes in adhesion culture, showing significantly higher levels of early stage marker AP2 for the DPSC compared to iPSC. To provide better environment for NCSC gene expression, suspension method was performed, which induced the formation of neurospheres. The results showed neurosphere formation after few days, obtaining the peak of NCSC marker expression after 4 days, showing overexpression of AP2, p75 and CHD7 markers, confirming the formation of NCSC like cells. Furthermore, pluripotent markers Oct4, Nanog and Sox2 were as well upregulated in suspension culture. Neurospheres were then directly cultured in CEC conditioned medium for the second differentiation, showing the conversion of DPSC into polygonal like cells expressing higher levels of ZO-1, ATP1A1, COL4A2 and COL8A1 markers, providing proof of the successful conversion into CEC. Conclusions Therefore, our findings demonstrate that patient-derived dental pulp stem cells represent an autologous cell source for corneal endothelial regeneration that avoids actual transplantation limitations as well as reprogramming techniques.