Viral-free generation and characterization of a human induced pluripotent stem cell line from dermal fibroblasts

Peripheral dermal fibroblasts (DF) from a healthy 56 year old female were obtained from the Centre for Healthy Brain Ageing (CHeBA) Biobank, University of New South Wales, under the material transfer agreement with the University of Wollongong. DFs were reprogrammed via mRNA-delivered transcription factors into induced pluripotent stem cells (iPSCs). The generated iPSCs were confirmed to be pluripotent, capable of three germ layer differentiation and are thus a useful resource for creating iPSC-derived healthy human cells of any lineage

The mRNA-based reprogramming of fibroblasts from a SOD1\u3csup\u3eE101G\u3c/sup\u3e familial amyotrophic lateral sclerosis patient to induced pluripotent stem cell line UOWi007

2020 The Authors Dermal fibroblasts were donated by a 43 year old male patient with clinically diagnosed familial amyotrophic lateral sclerosis (ALS), carrying the SOD1E101G mutation. The induced pluripotent stem cell (iPSC) line UOWi007-A was generated using repeated mRNA transfections for pluripotency transcription factors Oct4, Klf4, Sox2, c-Myc, Lin28 and Nanog. The iPSCs carried the SOD1E101G genotype and had a normal karyotype, expressed expected pluripotency markers and were capable of in vitro differentiation into endodermal, mesodermal and ectodermal lineages. This iPSC line may be useful for investigating familial ALS resulting from a SOD1 E101G mutation

The Ubiquitin Proteasome System Is a Key Regulator of Pluripotent Stem Cell Survival and Motor Neuron Differentiation

The ubiquitin proteasome system (UPS) plays an important role in regulating numerous cellular processes, and a dysfunctional UPS is thought to contribute to motor neuron disease. Consequently, we sought to map the changing ubiquitome in human iPSCs during their pluripotent stage and following differentiation to motor neurons. Ubiquitinomics analysis identified that spliceosomal and ribosomal proteins were more ubiquitylated in pluripotent stem cells, whilst proteins involved in fatty acid metabolism and the cytoskeleton were specifically ubiquitylated in the motor neurons. The UPS regulator, ubiquitin-like modifier activating enzyme 1 (UBA1), was increased 36-fold in the ubiquitome of motor neurons compared to pluripotent stem cells. Thus, we further investigated the functional consequences of inhibiting the UPS and UBA1 on motor neurons. The proteasome inhibitor MG132, or the UBA1-specific inhibitor PYR41, significantly decreased the viability of motor neurons. Consistent with a role of the UPS in maintaining the cytoskeleton and regulating motor neuron differentiation, UBA1 inhibition also reduced neurite length. Pluripotent stem cells were extremely sensitive to MG132, showing toxicity at nanomolar concentrations. The motor neurons were more resilient to MG132 than pluripotent stem cells but demonstrated higher sensitivity than fibroblasts. Together, this data highlights the important regulatory role of the UPS in pluripotent stem cell survival and motor neuron differentiation

Prospects for Food Fermentation in South-East Asia, Topics From the Tropical Fermentation and Biotechnology Network at the End of the AsiFood Erasmus+Project

Fermentation has been used for centuries to produce food in South-East Asia and some foods of this region are famous in the whole world. However, in the twenty first century, issues like food safety and quality must be addressed in a world changing from local business to globalization. In Western countries, the answer to these questions has been made through hygienisation, generalization of the use of starters, specialization of agriculture and use of long-distance transportation. This may have resulted in a loss in the taste and typicity of the products, in an extensive use of antibiotics and other chemicals and eventually, in a loss in the confidence of consumers to the products. The challenges awaiting fermentation in South-East Asia are thus to improve safety and quality in a sustainable system producing tasty and typical fermented products and valorising by-products. At the end of the “AsiFood Erasmus+ project” (www.asifood.org), the goal of this paper is to present and discuss these challenges as addressed by the Tropical Fermentation Network, a group of researchers from universities, research centers and companies in Asia and Europe. This paper presents current actions and prospects on hygienic, environmental, sensorial and nutritional qualities of traditional fermented food including screening of functional bacteria and starters, food safety strategies, research for new antimicrobial compounds, development of more sustainable fermentations and valorisation of by-products. A specificity of this network is also the multidisciplinary approach dealing with microbiology, food, chemical, sensorial, and genetic analyses, biotechnology, food supply chain, consumers and ethnology

Modelling neuronal excitability changes in ALS using iPSC-derived motor neurons and astrocytes

Amyotrophic lateral sclerosis (ALS) is the most severe form of Motor Neurone Disease. This fatal neurodegenerative disease causes the deterioration of the motor system leading to progressive paralysis. One of the earliest clinical observations in ALS patients is hyperexcitability of motor neurons in the motor cortex and spinal cord, prior to hypoexcitability and deterioration of motor function. The overarching objective of this thesis was to use induced pluripotent stem cells (iPSCs) from ALS patients to investigate electrophysiological changes to motor neurons, which may impact ALS progression. Firstly, the differentiation conditions of iPSC-derived motor neurons were optimised to improve the yield of electrophysiologically active motor neurons. Whole-cell patch clamping revealed that neuronal Na+ and K+ currents increased by more than 3-fold with the optimised culture conditions. Moreover, the proportion of repetitively firing neurons increased from less than 5% to approximately 75%. Secondly, using these improved culture conditions, the electrophysiological properties of CCNFS621G motor neurons were compared to CRISPR/Cas 9 generated isogenic control cell lines. The CCNFS621G motor neurons showed 3-fold increase in repetitively firing neurons compared to control motor neurons. This was further accompanied with a significant increase in Na+ and K+ currents. Together this suggests that the CCNFS621G mutation alters the electrophysiological properties of motor neurons leading to neuronal hyperexcitability. Finally, iPSC-derived motor neuron and astrocyte co-cultures were used to investigate the effect of ALS astrocytes on motor neuron excitability. ALS-derived astrocytes caused a loss of neuronal firing in both ALS and control motor neurons. Moreover, Na+ and K+ currents were reduced by up to 55% and 30%, respectively. Together these findings showed that the addition of ALS astrocytes induced hypoexcitability in ALS, as well as control motor neurons. This suggests that ALS astrocytes could be involved in the transition from motor neuron hyperexcitability to hypoexcitability. Overall the work presented in this thesis showed that the CCNFS621G mutation causes hyperexcitability in iPSC-derived motor neurons. However, this hyperexcitability phenotype was lost in the presence of ALS astrocytes. Thus, this work highlights that the cellular crosstalk between motor neurons and astrocytes plays a significant role in altering intrinsic neuronal excitability, which could impact ALS progression

Common pitfalls of stem cell differentiation: a guide to improving protocols for neurodegenerative disease models and research

Induced pluripotent stem cells and embryonic stem cells have revolutionized cellular neuroscience, providing the opportunity to model neurological diseases and test potential therapeutics in a pre-clinical setting. The power of these models has been widely discussed, but the potential pitfalls of stem cell differentiation in this research are less well described. We have analyzed the literature that describes differentiation of human pluripotent stem cells into three neural cell types that are commonly used to study diseases, including forebrain cholinergic neurons for Alzheimer\u27s disease, midbrain dopaminergic neurons for Parkinson\u27s disease and cortical astrocytes for neurodegenerative and psychiatric disorders. Published protocols for differentiation vary widely in the reported efficiency of target cell generation. Additionally, characterization of the cells by expression profile and functionality differs between studies and is often insufficient, leading to highly variable protocol outcomes. We have synthesized this information into a simple methodology that can be followed when performing or assessing differentiation techniques. Finally we propose three considerations for future research, including the use of physiological O2 conditions, three-dimensional co-culture systems and microfluidics to control feeding cycles and growth factor gradients. Following these guidelines will help researchers to ensure that robust and meaningful data is generated, enabling the full potential of stem cell differentiation for disease modeling and regenerative medicine

Impairments in motor neurons, interneurons and astrocytes contribute to hyperexcitability in ALS : underlying mechanisms and paths to therapy

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the loss of motor neurons leading to progressive paralysis and death. Using transcranial magnetic stimulation (TMS) and nerve excitability tests, several clinical studies have identified that cortical and peripheral hyperexcitability are among the earliest pathologies observed in ALS patients. The changes in the electrophysiological properties of motor neurons have been identified in both sporadic and familial ALS patients, despite the diverse etiology of the disease. The mechanisms behind the change in neuronal signalling are not well understood, though current findings implicate intrinsic changes in motor neurons and dysfunction of cells critical in regulating motor neuronal excitability, such as astrocytes and interneurons. Alterations in ion channel expression and/or function in motor neurons has been associated with changes in cortical and peripheral nerve excitability. In addition to these intrinsic changes in motor neurons, inhibitory signalling through GABAergic interneurons is also impaired in ALS, likely contributing to increased neuronal excitability. Astrocytes have also recently been implicated in increasing neuronal excitability in ALS by failing to adequately regulate glutamate levels and extracellular K+ concentration at the synaptic cleft. As hyperexcitability is a common and early feature of ALS, it offers a therapeutic and diagnostic target. Thus, understanding the underlying pathways and mechanisms leading to hyperexcitability in ALS offers crucial insight for future development of ALS treatments

Impairments in Motor Neurons, Interneurons and Astrocytes Contribute to Hyperexcitability in ALS: Underlying Mechanisms and Paths to Therapy

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the loss of motor neurons leading to progressive paralysis and death. Using transcranial magnetic stimulation (TMS) and nerve excitability tests, several clinical studies have identified that cortical and peripheral hyperexcitability are among the earliest pathologies observed in ALS patients. The changes in the electrophysiological properties of motor neurons have been identified in both sporadic and familial ALS patients, despite the diverse etiology of the disease. The mechanisms behind the change in neuronal signalling are not well understood, though current findings implicate intrinsic changes in motor neurons and dysfunction of cells critical in regulating motor neuronal excitability, such as astrocytes and interneurons. Alterations in ion channel expression and/or function in motor neurons has been associated with changes in cortical and peripheral nerve excitability. In addition to these intrinsic changes in motor neurons, inhibitory signalling through GABAergic interneurons is also impaired in ALS, likely contributing to increased neuronal excitability. Astrocytes have also recently been implicated in increasing neuronal excitability in ALS by failing to adequately regulate glutamate levels and extracellular K+ concentration at the synaptic cleft. As hyperexcitability is a common and early feature of ALS, it offers a therapeutic and diagnostic target. Thus, understanding the underlying pathways and mechanisms leading to hyperexcitability in ALS offers crucial insight for future development of ALS treatments

Preparation and characterization of chitosan/

Biofilm preservation has become a topic of interest among many scientists. A recent study involved the production of chitosan-based biofilms containing varying amounts of aloe vera gel (5%, 10% and 15% w/w). Evaluation of film formation, water absorption, swelling ratio, solubility, antibacterial ability, colouration, and biodegradability indicated that chitosan film added with 10% aloe vera (CS-10%AV) had the best properties. The study also found that using CS-A10 film to preserve tomatoes helped maintain the fruit’s colour and shape for longer while retaining vitamin C and acids for an extended period