Interleukin-7 enhances in vitro development and blastocyst quality in porcine parthenogenetic embryos

Interleukin-7 (IL-7), a vital factor that affects cell development, proliferation, and survival, plays an important role in oocyte maturation. However, its role in embryonic development remains unknown. Therefore, in this study, we aimed to investigate the effects of IL-7 supplementation on in vitro culture (IVC) of porcine embryos after parthenogenetic activation (PA) based on characteristics such as cleavage, blastocyst formation rate, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels in cleaved embryos, total cell number, apoptosis rate, and cell lineage specification in blastocysts. Immunofluorescence revealed that IL-7 and its receptor, IL-7Rα (IL-7R) localized in the cytoplasm of porcine parthenote embryos. By supplementing the IVC medium (PZM5) with various concentrations of IL-7, an optimal concentration that enhanced embryonic development, promoted intracellular GSH, and decreased ROS levels in the cleavage stage during porcine embryo IVC was determined. Investigation of mRNA expression patterns via qRT-PCR suggested that IL-7 possibly regulated maternal mRNA clearance and zygotic genome activation. Furthermore, IL-7 supplementation reduced blastocyst apoptosis, enhanced the expression of the inner cell mass marker SOX2, and phosphorylated STAT5 levels in the blastocysts. Moreover, it altered the transcription patterns of genes that regulate apoptosis, IL-7 signaling, and development. Thus, we demonstrated the localization of IL-7 and IL-7R in porcine preimplantation embryos in vitro for the first time. Furthermore, we suggest that IL-7 supplementation can be employed to enhance embryonic development and blastocyst quality based on the activation of the transcripts of genes that are involved in developmental competence and IL-7 signaling during in vitro porcine embryo development following PA

Modeling Neural Crest Induction, Melanocyte Specification, and Disease-Related Pigmentation Defects in hESCs and Patient-Specific iPSCs

Melanocytes are pigment-producing cells of neural crest (NC) origin that are responsible for protecting the skin against UV irradiation. Pluripotent stem cell (PSC) technology offers a promising approach for studying human melanocyte development and disease. Here, we report that timed exposure to activators of WNT, BMP, and EDN3 signaling triggers the sequential induction of NC and melanocyte precursor fates under dual-SMAD-inhibition conditions. Using a SOX10::GFP human embryonic stem cell (hESC) reporter line, we demonstrate that the temporal onset of WNT activation is particularly critical for human NC induction. Subsequent maturation of hESC-derived melanocytes yields pure populations that match the molecular and functional properties of adult melanocytes. Melanocytes from Hermansky-Pudlak syndrome and Chediak-Higashi syndrome patient-specific induced PSCs (iPSCs) faithfully reproduce the ultrastructural features of disease-associated pigmentation defects. Our data define a highly specific requirement for WNT signaling during NC induction and enable the generation of pure populations of human iPSC-derived melanocytes for faithful modeling of pigmentation disorders

Neuronal Differentiation of Human Embryonic Stem Cell-Derived Neural Crest Stem Cells by Pulsed Electrical Field

This work surveyed the potential of using exogenous pulsed electrical field stimuli to guide the differentiation of human embryonic stem cell (hESC) derived-neural crest stem cells (NCSCs) towards their neuronal lineage. A vertical electrode bioreactor able to vary the parameters of direct current pulse frequency, width and intensity was fabricated for this purpose. Dendritic morphology was observed for hNCSCs cultured on cathodes subject to 1 Hz, 50 ms pulse at 150mV/mm and 200mV/mm for 24 hours. The morphological phenomena were observed in a dosage and polarity orientation dependent manner; however, significant apoptosis was observed post stimulation. hNCSCs and hESCs were then cultured on cathodes and subject to 1Hz, 50ms pulse at 150mV/mm and 200mV/mm for 1.5 hours/day and 3 hours/day for 8 days. The expression of neuron-specific class III beta tubulin (Tuj1) was observed through immunofluorescence post stimulation and the expression levels of Tuj1 were qualitatively higher for the stimulated hNCSCs and hESCs as compared to the controls. Apoptosis was only observed in hNCSCs subject to the harshest condition. Ethylene glycol tetraacetic acid (EGTA) was used to sequester extracellular Ca2+ in the media during the stimulation of hNCSCs on cathodes subject to 1Hz, 50ms pulse at 200mV/mm for 24 hours. As EGTA concentrations were increased from 0mM to 2mM, dendritic morphology was reduced. Thus, the morphological phenomena were correlated to the available free extracellular calcium concentrations. Further investigation into the intracellular calcium concentrations in stimulated hNCSCs and hESCs was accomplished through the Fluo-4 assay. hNCSCs and hESCs were stimulated on cathodes subject to 2Hz, 50ms pulse at 200mV/mm for 5 hours. Increased chronic levels of intracellular calcium were observed for hNCSC and hESC samples post stimulation

Evaluation of developmental toxicants and signaling pathways in a functional test based on the migration of human neural crest cells

BACKGROUND: Information on the potential developmental toxicity (DT) of the majority of chemicals is scarce, and test capacities for further animal-based testing are limited. Therefore, new approaches with higher throughput are required. A screening strategy based on the use of relevant human cell types has been proposed by the EPA and others. As impaired neural crest (NC) function is one of the known causes for teratologic effects, testing of toxicant effects on NC is desirable for a DT test battery.OBJECTIVE: To develop a robust and widely applicable human-relevant NC function assay, allowing sensitive screening of environmental toxicants, and a definition of toxicity pathways.METHODS: We generated NC cells from human embryonic stem cells, and after establishing a migration assay of NC (MINC), we tested environmental toxicants as well as inhibitors of physiological signal transduction pathways.RESULTS: Methylmercury (50 nM), valproic acid (> 10 µM) and Lead-acetate (1 µM) affected migration of NC more potently than migration of other cell types. The MINC assay correctly identified the neural crest toxicants triadimefon and triadimenol, additionally it showed different sensitivities to various organic and inorganic mercury compounds. Applying classic pharmacologic inhibitors and large-scale microarray gene expression profiling, we found several signaling pathways that are relevant for the migration of NC in the MINC.CONCLUSIONS: The MINC assay faithfully models human NC migration, and reveals impairment of this function by developmental toxicants with good sensitivity and specificity

Inhibition of the Combinatorial Signaling of Transforming Growth Factor-Beta and NOTCH Promotes Myotube Formation of Human Pluripotent Stem Cell-Derived Skeletal Muscle Progenitor Cells

Understanding the signaling pathways that regulate the final differentiation of human myoblasts is essential for successful cell transplantation and drug screening for the treatment of muscular dystrophy. In an effort to improve myotube formation from hiPSC-derived myoblasts, we validated a collection of 13 small molecules in a newly established in vitro screening platform for the assessment of myotube formation. The analysis of myotube formation as measured by the fusion index showed that the combinational inhibition of the TGFβ signaling with NOTCH signaling enhances the ability of multi-nucleated myotube production. Combinational treatment of inhibitors for TGFβ and NOTCH signaling pathways improved myotube formation in a dose-dependent manner. This effect was achieved by inhibiting the combinatorial mechanism of signaling. The combination treatment of small molecules effective in inducing multinucleated myotubes was validated in healthy human primary myoblasts. In addition, it was also applied to DMD patient iPSC-derived myoblasts to enhance the generation of multinucleated myotubes