Extensive Nuclear Reprogramming Underlies Lineage Conversion into Functional Trophoblast Stem-like Cells

SummaryInduced pluripotent stem cells (iPSCs) undergo extensive nuclear reprogramming and are generally indistinguishable from embryonic stem cells (ESCs) in their functional capacity and transcriptome and DNA methylation profiles. However, direct conversion of cells from one lineage to another often yields incompletely reprogrammed, functionally compromised cells, raising the question of whether pluripotency is required to achieve a high degree of nuclear reprogramming. Here, we show that transient expression of Gata3, Eomes, and Tfap2c in mouse fibroblasts induces stable, transgene-independent trophoblast stem-like cells (iTSCs). iTSCs possess transcriptional profiles highly similar to blastocyst-derived TSCs, with comparable methylation and H3K27ac patterns and genome-wide H2A.X deposition. iTSCs generate trophoectodermal lineages upon differentiation, form hemorrhagic lesions, and contribute to developing placentas in chimera assays, indicating a high degree of nuclear reprogramming, with no evidence of passage through a transient pluripotent state. Together, these data demonstrate that extensive nuclear reprogramming can be achieved independently of pluripotency

Concise Review: Stem Cell Models of SCN1A-Related Encephalopathies—Current Perspective and Future Therapies

Mutations in the SCN1A gene can cause a variety of phenotypes, ranging from mild forms, such as febrile seizures and generalized epilepsy with febrile seizures plus, to severe, such as Dravet and non-Dravet developmental epileptic encephalopathies. Until now, more than two thousand pathogenic variants of the SCN1A gene have been identified and different pathogenic mechanisms (loss vs. gain of function) described, but the precise molecular mechanisms responsible for the deficits exhibited by patients are not fully elucidated. Additionally, the phenotypic variability proves the involvement of other genetic factors in its final expression. This is the reason why animal models and cell line models used to explore the molecular pathology of SCN1A-related disorders are only of limited use. The results of studies based on such models cannot be directly translated to affected individuals because they do not address each patient’s unique genetic background. The generation of functional neurons and glia for patient-derived iPSCs, together with the generation of isogenic controls using CRISPR/Cas technology, and finally, the 3D brain organoid models, seem to be a good way to solve this problem. Here, we review SCN1A-related encephalopathies, as well as the stem cell models used to explore their molecular basis

DataSheet1_Electrochemical Studies of the Cycloaddition Activity of Bismuth(III) Acetylides Towards Organic Azides Under Copper(I)-Catalyzed Conditions.pdf

Time-dependent monitoring of the reactive intermediates provides valuable information about the mechanism of a synthetic transformation. However, the process frequently involves intermediates with short lifetimes that significantly challenge the accessibility of the desired kinetic data. We report in situ cyclic voltammetry (CV) and nuclear magnetic resonance (NMR) spectroscopy studies of the cycloaddition reaction of organobismuth(III) compounds with organic azides under the copper(I)-catalyzed conditions. A series of bismuth(III) acetylides carrying diphenyl sulfone scaffolds have been synthesized to study the underlying electronic and steric effects of the tethered moieties capable of transannular oxygen O···Bi interactions and para-functionality of the parent phenylacetylene backbones. While belonging to the family of copper-catalyzed azide-alkyne cycloaddition reactions, the reaction yielding 5-bismuth(III)-triazolide is the sole example of a complex catalytic transformation that features activity of bismuth(III) acetylides towards organic azides under copper(I)-catalyzed conditions. Stepwise continuous monitoring of the copper(I)/copper(0) redox activity of the copper(I) catalyst by cyclic voltammetry provided novel insights into the complex catalytic cycle of the bismuth(III)-triazolide formation. From CV-derived kinetic data, reaction rate parameters of the bismuth(III) acetylides coordination to the copper(I) catalyst (KA) and equilibrium concentration of the copper species [cat]eq. are compared with the overall 5-bismuth(III)-triazolide formation rate constant kobs obtained by 1H-NMR kinetic analysis.</p

Safety of GMP-compliant iPSC lines generated by Sendai virus transduction is dependent upon clone identity and sex of the donor

Human induced pluripotent stem cells (hiPSCs) are a potential source of somatic cells for cell therapies due to their ability to self-renew and differentiate into various cells of the body. To date, the clinical application of hiPSCs has been limited due to safety issues. The present study aims to standardize the safety procedure of the derivation of GMP-compliant induced pluripotent stem cell (iPSC) lines from human fibroblasts. The hiPSC lines were generated using the nonintegrative Sendai virus method to incorporate Yamanaka reprogramming factors (OCT3/4, SOX2, KLF4 and c-MYC) into cells. A constant temperature was maintained during the cell culture, including all stages of the culture after transduction with Sendai virus. Pluripotency was proved in six independently generated hiPSC lines from adult female (47 years old) and male (57 years old) donors’ derived fibroblasts via alkaline phosphatase live (ALP) staining, qPCR, and immunocytochemistry. The hiPSC lines showed a gradual decrease in the presence of the virus with each subsequent passage, and this reduction was specific to the hiPSC line. The frequency and probability of chromosomal aberrations in hiPSCs were dependent on both the iPSC clone identity and sex of the donor. In summary, the generation of hiPSC for clinical applications requires safety standards application (biosafety protocol, quality control of hiPSC lines, viral and genetic integrity screening) from the first stages of the clonal selection of hiPSC from the same donor

The Generation of Human iPSC Lines from Three Individuals with Dravet Syndrome and Characterization of Neural Differentiation Markers in iPSC-Derived Ventral Forebrain Organoid Model

Dravet syndrome (DRVT) is a rare form of neurodevelopmental disorder with a high risk of sudden unexpected death in epilepsy (SUDEP), caused mainly (>80% cases) by mutations in the SCN1A gene, coding the Nav1.1 protein (alfa-subunit of voltage-sensitive sodium channel). Mutations in SCN1A are linked to heterogenous epileptic phenotypes of various types, severity, and patient prognosis. Here we generated iPSC lines from fibroblasts obtained from three individuals affected with DRVT carrying distinct mutations in the SCN1A gene (nonsense mutation p.Ser1516*, missense mutation p.Arg1596His, and splicing mutation c.2589+2dupT). The iPSC lines, generated with the non-integrative approach, retained the distinct SCN1A gene mutation of the donor fibroblasts and were characterized by confirming the expression of the pluripotency markers, the three-germ layer differentiation potential, the absence of exogenous vector expression, and a normal karyotype. The generated iPSC lines were used to establish ventral forebrain organoids, the most affected type of neurons in the pathology of DRVT. The DRVT organoid model will provide an additional resource for deciphering the pathology behind Nav1.1 haploinsufficiency and drug screening to remediate the functional deficits associated with the disease