Epithelial cadherin regulates transition between the naïve and primed pluripotent states in mouse embryonic stem cells.

Inhibition of E-cad in mouse embryonic stem cells (mESCs) leads to a switch from LIF-BMP to Activin/Nodal-dependent pluripotency, consistent with transition from a naïve to primed pluripotent phenotype. We have used both genetic ablation and steric inhibition of E-cad function in mESCs to assess alterations to phenotype using quantitative mass spectrometry analysis, network models, and functional assays. Proteomic analyses revealed that one third of detected proteins were altered in E-cad null mESCs (Ecad-/- mESCs) compared to wild type (624 proteins were downregulated and 705 were proteins upregulated). Network pathway analysis and subsequent cellular flux assays confirmed a metabolic shift from oxidative phosphorylation (OXPHOS) to aerobic glycolysis, specifically through mitochondrial complex III downregulation and hypoxia inducible factor 1a target upregulation. Central to this was the transcriptional coactivator EP300. E-cad is a well-known tumor suppressor, its downregulation during cancer initiation and metastasis can be linked to the metabolic switch known as Warburg effect. This study highlights a phenomena found in both primed pluripotent state and cancer stemness and links it to loss of E-cad. Data are available via ProteomeXchange with identifier PXD012679.

Safe and stable generation of induced pluripotent stem cells using doggybone DNA vectors.

The application of induced pluripotent stem cells (iPSCs) in advanced therapies is increasing at pace, but concerns remain over their clinical safety profile. We report the first-ever application of doggybone DNA (dbDNA) vectors to generate human iPSCs. dbDNA vectors are closed-capped linear double-stranded DNA gene expression cassettes that contain no bacterial DNA and are amplified by a chemically defined, current good manufacturing practice (cGMP)-compliant methodology. We achieved comparable iPSC reprogramming efficiencies using transiently expressing dbDNA vectors with the same iPSC reprogramming coding sequences as the state-of-the-art OriP/EBNA1 episomal vectors but, crucially, in the absence of p53 shRNA repression. Moreover, persistent expression of EBNA1 from bacterially derived episomes resulted in stimulation of the interferon response, elevated DNA damage, and increased spontaneous differentiation. These cellular activities were diminished or absent in dbDNA-iPSCs, resulting in lines with a greater stability and safety potential for cell therapy.

Cellular models of Batten disease.

The Neuronal Ceroid Lipofuscinoses (NCL), otherwise known as Batten disease, are a group of neurodegenerative diseases caused by mutations in 13 known genes. All except one NCL is autosomal recessive in inheritance, with similar aetiology and characterised by the accumulation of autofluorescent storage material in the lysosomes of cells. Age of onset and the rate of progression vary between the NCLs. They are collectively one of the most common lysosomal storage diseases, but the enigma remains of how genetically distinct diseases result in such remarkably similar pathogenesis. Much has been learnt from cellular studies about the function of the proteins encoded by the affected genes. Such research has utilised primitive unicellular models such as yeast and amoeba containing gene orthologues, cells derived from naturally occurring (sheep) and genetically engineered (mouse) animal models or patient-derived cells. Most recently, patient-derived induced pluripotent stem cell (iPSC) lines have been differentiated into neural cell-types to study molecular pathogenesis in the cells most profoundly affected by disease. Here, we review how cell models have informed much of the biochemical understanding of the NCLs and how more complex models are being used to further this understanding and potentially act as platforms for therapeutic efficacy studies in the future.

Continual Conscious Bioluminescent Imaging in Freely Moving Mice.

In vivo bioluminescent imaging allows the detection of reporter gene expression in rodents in real time. Here we describe a novel technology whereby we can generate somatotransgenic rodents with the use of a viral vector carrying a luciferase transgene. We are able to achieve long term luciferase expression by a single injection of lentiviral or adeno-associated virus vectors to newborn mice. Further, we describe whole body bioluminescence imaging of conscious mice in a noninvasive manner, thus enforcing the 3R's (replacement, reduction, and refinement) of biomedical animal research.

A functional human motor unit platform engineered from human embryonic stem cells and immortalized skeletal myoblasts.

BACKGROUND: Although considerable research on neuromuscular junctions (NMJs) has been conducted, the prospect of in vivo NMJ studies is limited and these studies are challenging to implement. Therefore, there is a clear unmet need to develop a feasible, robust, and physiologically relevant in vitro NMJ model. OBJECTIVE: We aimed to establish a novel functional human NMJs platform, which is serum and neural complex media/neural growth factor-free, using human immortalized myoblasts and human embryonic stem cells (hESCs)-derived neural progenitor cells (NPCs) that can be used to understand the mechanisms of NMJ development and degeneration. METHODS: Immortalized human myoblasts were co-cultured with hESCs derived committed NPCs. Over the course of the 7 days myoblasts differentiated into myotubes and NPCs differentiated into motor neurons. RESULTS: Neuronal axon sprouting branched to form multiple NMJ innervation sites along the myotubes and the myotubes showed extensive, spontaneous contractile activity. Choline acetyltransferase and ßIII-tubulin immunostaining confirmed that the NPCs had matured into cholinergic motor neurons. Postsynaptic site of NMJs was further characterized by staining dihydropyridine receptors, ryanodine receptors, and acetylcholine receptors by α-bungarotoxin. CONCLUSION: We established a functional human motor unit platform for in vitro investigations. Thus, this co-culture system can be used as a novel platform for 1) drug discovery in the treatment of neuromuscular disorders, 2) deciphering vital features of NMJ formation, regulation, maintenance, and repair, and 3) exploring neuromuscular diseases, age-associated degeneration of the NMJ, muscle aging, and diabetic neuropathy and myopathy.

NF-κB Activity Initiates Human ESC-Derived Neural Progenitor Cell Differentiation by Inducing a Metabolic Maturation Program.

Human neural development begins at embryonic day 19 and marks the beginning of organogenesis. Neural stem cells in the neural tube undergo profound functional, morphological, and metabolic changes during neural specification, coordinated by a combination of exogenous and endogenous cues. The temporal cell signaling activities that mediate this process, during development and in the postnatal brain, are incompletely understood. We have applied gene expression studies and transcription factor-activated reporter lentiviruses during in vitro neural specification of human pluripotent stem cells. We show that nuclear factor κB orchestrates a multi-faceted metabolic program necessary for the maturation of neural progenitor cells during neurogenesis.

Bioluminescence Monitoring of Promoter Activity In Vitro and In Vivo.

The application of luciferase reporter genes to provide quantitative outputs for the activation of promoters is a well-established technique in molecular biology. Luciferase catalyzes an enzymatic reaction, which in the presence of the substrate luciferin produces photons of light relative to its molar concentration. The luciferase transgene can be genetically inserted at the first intron of a target gene to act as a surrogate for the gene's endogenous expression in cells and transgenic mice. Alternatively, promoter sequences can be excised and/or amplified from genomic sources or constructed de novo and cloned upstream of luciferase in an expression cassette transfected into cells. More recently, the development of synthetic promoters where the essential components of an RNA polymerase binding site and transcriptional start site are fused with various upstream regulatory sequences are being applied to drive reporter gene expression. We have developed a high-throughput cloning strategy to develop lentiviral luciferase reporters driven by transcription factor activated synthetic promoters. Lentiviruses integrate their payload cassette into the host cell genome, thereby facilitating the study of gene expression not only in the transduced cells but also within all subsequent daughter cells. In this manuscript we describe the design, vector construction, lentiviral transduction, and luciferase quantitation of transcription factor activated reporters (TFARs) in vitro and in vivo.

NRF2 Orchestrates the Metabolic Shift during Induced Pluripotent Stem Cell Reprogramming.

The potential of induced pluripotent stem cells (iPSCs) in disease modeling and regenerative medicine is vast, but current methodologies remain inefficient. Understanding the cellular mechanisms underlying iPSC reprogramming, such as the metabolic shift from oxidative to glycolytic energy production, is key to improving its efficiency. We have developed a lentiviral reporter system to assay longitudinal changes in cell signaling and transcription factor activity in living cells throughout iPSC reprogramming of human dermal fibroblasts. We reveal early NF-κB, AP-1, and NRF2 transcription factor activation prior to a temporal peak in hypoxia inducible factor α (HIFα) activity. Mechanistically, we show that an early burst in oxidative phosphorylation and elevated reactive oxygen species generation mediates increased NRF2 activity, which in turn initiates the HIFα-mediated glycolytic shift and may modulate glucose redistribution to the pentose phosphate pathway. Critically, inhibition of NRF2 by KEAP1 overexpression compromises metabolic reprogramming and results in reduced efficiency of iPSC colony formation.