Fine Tuning of Hepatocyte Differentiation from Human Embryonic Stem Cells: Growth Factor vs. Small Molecule-Based Approaches

Human embryonic stem cells (hESCs) are being utilized in diverse areas of studies such as development and disease modeling, cell replacement therapy, or drug toxicity testing because of their potential to be differentiated into any cell type in the body. The directed differentiation of hESCs into hepatocytes could provide an invaluable source of liver cells for various liver-based applications. Therefore, several protocols have been established in the past for hESC-hepatocyte differentiation based on the knowledge of signaling pathways and growth factors involved in different stages of embryonic hepatogenesis. Although successful derivation of hepatocytes has been achieved through these protocols, the efficiency is not always ideal. Herein, we have tested several combinations of published protocols, for example, growth factor vs. small molecule and different time durations of treatment for definitive endoderm (DE) induction and further hepatocyte differentiation to develop an efficient DE induction and hepatocyte differentiation in a highly reproducible manner based on the stage-specific marker expression and functional analysis

Neuroprotective Effects of Thymol, a Dietary Monoterpene Against Dopaminergic Neurodegeneration in Rotenone-Induced Rat Model of Parkinson’s Disease

Parkinson’s disease (PD), a multifactorial movement disorder that involves progressive degeneration of the nigrostriatal system affecting the movement ability of the patient. Oxidative stress and neuroinflammation both are shown to be involved in the etiopathogenesis of PD. The aim of this study was to evaluate the therapeutic potential of thymol, a dietary monoterpene phenol in rotenone (ROT)-induced neurodegeneration in rats that precisely mimics PD in humans. Male Wistar rats were injected ROT at a dose of 2.5 mg/kg body weight for 4 weeks, to induce PD. Thymol was co-administered for 4 weeks at a dose of 50 mg/kg body weight, 30 min prior to ROT injection. The markers of dopaminergic neurodegeneration, oxidative stress and inflammation were estimated using biochemical assays, enzyme-linked immunosorbent assay, western blotting and immunocytochemistry. ROT challenge increased the oxidative stress markers, inflammatory enzymes and cytokines as well as caused significant damage to nigrostriatal dopaminergic system of the brain. Thymol treatment in ROT challenged rats appears to significantly attenuate dopaminergic neuronal loss, oxidative stress and inflammation. The present study showed protective effects of thymol in ROT-induced neurotoxicity and neurodegeneration mediated by preservation of endogenous antioxidant defense networks and attenuation of inflammatory mediators including cytokines and enzymes

Saturated fatty acid regulated lncRNA dataset during in vitro human embryonic neurogenesis

Human embryonic stem cells (hESCs) were used as a model of embryonic neurogenesis to identify the effect of excess fat uptake on neurodevelopment (Ardah et al., 2018). Herein, by directed differentiation of hESCs into neurons using established protocols, this data was generated for expression profiles of select lncRNAs during in vitro embryonic neurogenesis and their differential expression due to excess fat (palmitate) uptake. The undifferentiated hESCs were treated with 250 µM palmitate after identifying it as the highest concentration which is non-toxic to these cells. The palmitate treated hESCs were differentiated towards neurons keeping the levels of palmitate consistent throughout the differentiation process and fat uptake was confirmed by Oil Red O staining. The expression analysis of lncRNAs was performed by RT-qPCR on vehicle control and palmitate treated cells from 4 stages of differentiation, D0 (undifferentiated hESCs), D12 (neural stem cells), D44 (neural progenitors) and D70 (neurons) using lncRNAs array plates from Arraystar Inc. which contains 372 functionally identified lncRNAs found to be associated with lipid metabolism and other pathways (Cat# AS-NR-004)

The TATA-Box Sequence in the Basal Promoter Contributes to Determining Light-Dependent Gene Expression in Plants

A prototype 13-bp TATA-box sequence, TCACTATATATAG, was mutated at each nucleotide position and examined for its function in the core promoter. Specific nucleotides in the first TATA, the second TATA, as well as the flanking sequences influenced promoter function in transient transformation of tobacco (Nicotiana tabacum var Petit Havana) leaves. The effect of a given mutation on reporter gene expression in light versus dark was variable and sometimes contrasting. Some mutations, like T(7) or A(8)→C or G, completely inactivated the expression of the minimal promoter in light but not in dark. In general, the sequence requirement for dark expression was less stringent than that for light expression. The selective effect of TATA-box mutations on light versus dark expression was exerted on core promoter function in the chromatin-integrated state also. Even in the presence of an upstream light response activator element, TATA-box mutations influenced modulation of the promoter by light. An A at the eighth position was specifically involved in the red light response of the promoter. Selectivity in gene expression was associated with a high level of transcript initiation from a site that was not active in the dark. Nuclear proteins from dark- and light-grown seedlings showed that the sequence variation within the TATA-box governs the formation of alternative transcriptional complexes. The experiments give direct evidence for the role of a core TATA-box sequence in determining the level as well as selectivity of gene expression in plants

A T9G Mutation in the Prototype TATA-Box TCACTATATATAG Determines Nucleosome Formation and Synergy with Upstream Activator Sequences in Plant Promoters1[W]

We had earlier reported that mutations to G and C at the seventh and eighth positions in the prototype TATA-box TCACTATATATAG inhibited light-dependent activation of transcription from the promoter. In this study, we characterized mutations at the ninth position of the prototype TATA-box. Substitution of T at the ninth position with G or C enhanced transcription from the promoter in transgenic tobacco (Nicotiana tabacum) plants. The effect of T9G/C mutations was not light dependent, although the 9G/C TATA-box showed synergy with the light-responsive element (lre). However, the 9G/C mutants in the presence of lre failed to respond to phytochromes, sugar, and calcium signaling, in contrast to the prototype TATA-box with lre. The 9G/C mutation shifted the point of initiation of transcription, and transcription activation was dependent upon the type of activating element present upstream. The synergy in activation was noticed with lre and legumin activators but not with rbcS, Pcec, and PR-1a activators. The 9G mutation resulted in a micrococcal nuclease-sensitive region over the TATA-box, suggesting a nucleosome-free region, in contrast to the prototype promoter, which had a distinct nucleosome on the TATA-box. Thus, the transcriptional augmentation with mutation at the ninth position might be because of the loss of a repressive nucleosomal structure on the TATA-box. In agreement with our findings, the promoters containing TATAGATA as identified by genome-wide analysis of Arabidopsis (Arabidopsis thaliana) are not tightly repressed

Higher O-GlcNAc Levels Are Associated with Defects in Progenitor Proliferation and Premature Neuronal Differentiation during in-Vitro Human Embryonic Cortical Neurogenesis

The nutrient responsive O-GlcNAcylation is a dynamic post-translational protein modification found on several nucleocytoplasmic proteins. Previous studies have suggested that hyperglycemia induces the levels of total O-GlcNAcylation inside the cells. Hyperglycemia mediated increase in protein O-GlcNAcylation has been shown to be responsible for various pathologies including insulin resistance and Alzheimer's disease. Since maternal hyperglycemia during pregnancy is associated with adverse neurodevelopmental outcomes in the offspring, it is intriguing to identify the effect of increased protein O-GlcNAcylation on embryonic neurogenesis. Herein using human embryonic stem cells (hESCs) as model, we show that increased levels of total O-GlcNAc is associated with decreased neural progenitor proliferation and premature differentiation of cortical neurons, reduced AKT phosphorylation, increased apoptosis and defects in the expression of various regulators of embryonic corticogenesis. As defects in proliferation and differentiation during neurodevelopment are common features of various neurodevelopmental disorders, increased O-GlcNAcylation could be one mechanism responsible for defective neurodevelopmental outcomes in metabolically compromised pregnancies such as diabetes

Nutrient sensitive protein O-GlcNAcylation modulates the transcriptome through epigenetic mechanisms during embryonic neurogenesis

10.26508/lsa.202201385LIFE SCIENCE ALLIANCE58completedcomplete

Identification of early indicators of altered metabolism in normal development using a rodent model system

Although the existence of a close relationship between the early maternal developmental environment, fetal size at birth and the risk of developing disease in adulthood has been suggested, most studies, however, employed experimentally induced intrauterine growth restriction as a model to link this with later adult disease. Because embryonic size variation also occurs under normal growth and differentiation, elucidating the molecular mechanisms underlying these changes and their relevance to later adult disease risk becomes important. The birth weight of rat pups vary according to the uterine horn positions. Using birth weight as a marker, we compared two groups of rat pups – lower birth weight (LBW, 5th to 25th percentile) and average birth weight (ABW, 50th to 75th percentile) – using morphological, biochemical and molecular biology, and genetic techniques. Our results show that insulin metabolism, Pi3k/Akt and Pparγ signaling and the genes regulating growth and metabolism are significantly different in these groups. Methylation at the promoter of the InsII (Ins2) gene and DNA methyltransferase 1 in LBW pups are both increased. Additionally, the Dnmt1 repressor complex, which includes Hdac1, Rb (Rb1) and E2f1, was also upregulated in LBW pups. We conclude that the Dnmt1 repressor complex, which regulates the restriction point of the cell cycle, retards the rate at which cells traverse the G1 or G0 phase of the cell cycle in LBW pups, thereby slowing down growth. This regulatory mechanism mediated by Dnmt1 might contribute to the production of small-size pups and altered physiology and pathology in adult life