Study of living tissues has gained wide spread application in microscopy with the advent of fluorescent dyes and inverted microscopes dedicated to their viewing. A critical component is maintaining physiological conditions in media formulations, including pH and osmolarity, without contributing to background fluorescence. Most cells are grown in media formulations that use bicarbonate buffers, requiring the continuous presence of CO2 to maintain physiological pH. Just 15 minutes outside a CO2 incubator can change the pH from 7.4 to 8.1, and to 8.7 after an hour. [Read more…]
mRNAs are expression factors that mimic fully processed mRNA. Being the substrate for translation by ribosomes, mRNA expression factors are often preferred over viral vectors for cell reprogramming and iPS cell generation because of the absent risk of integration into the genome. Such RNA-induced pluripotent stem cells (RiPSCs described in 2010 by Warren et al.) are becoming more and more popular. 3 reasons might illustrate RiPSCs’ attractivty.
In 2010, a method for integration-free reprogramming by transfecting modified mRNA reprogramming factors was published (1, 2). The use of mRNA to induce reprogramming of somatic cells overcomes the inherent problems incurred by introducing viral vectors and/or integrating DNA to target cells. mRNA reprogramming factors are titratable and controllable with regards to the ratios, concentrations, and timing of factor expression. The mRNA reprogramming system also poses none of the biosafety risks surrounding viral and DNA-based systems. [Read more…]
It is widely accepted that data obtained from primary cells is not only desired, but, most relevant when trying to study physiological interactions. Experimentation in primary cells allows a deeper and more physiologically relevant view into the cellular function of proteins and enzymes that will allow the design of more specific drugs with meaningful and specific targets.
Chipman et al. (Dalhousie University, Halifax, Nova Scotia, Canada) have developed a new cell-based system “mimicking” in vitro Mature Neuromuscular Junction (NMJs). (1)
NMJs play an important role in the functionality of Motor neurons.
Their dysfunction is seen in the early stages of various Motor Neuron Diseases (MNDs) like the Amyotrophic Lateral Sclerosis (ALS), Spinal Muscular Atrophy (SMA), Primary Lateral Sclerosis (PLS), Progressive Muscular Atrophy (PMA)…
To meet these needs, Chipman et al. have created a clever co-culture system using Embryonic Stem Cell-derived Motor Neurons (ESCMNs) with primary myotubes. They also reported that NCAM-/- ESCMN/myofiber co-cultures exhibit the same phenotypes observed in NCAM-/- mice.
This work give hopes to study MNDs in vitro by using motor neurons differentiated from induced Pluripotent Stem cells (iPS) derived from individuals with ALS and SMA.
Want to know more about cell-based assays and co-culture systems?
(1) Chipman et al. “A Stem-Cell Based Bioassay to Critically Assess the Pathology of Dysfunctional Neuromuscular Junctions” (2014) PLOSone, March 13. DOI: 10.1371/journal.pone.0091643
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- Primary and iPS cells