Epidermal growth factor (EGF) is characterized by high affinity binding to various EGF receptors (EGFRs) and the production of mitogenic responses (Carpenter & Cohen). EGF promotes EGFR dimerization, resulting in activation of downstream pathways including PI3K, ERK1/2, JAK/STAT, β-catenin, and calcium signaling. EGF is secreted by the gut-associated salivary and Brunner’s glands, is found in a variety of body fluids, and stimulates cell proliferation and differentiation in rodent and neonatal human intestine (Wright et al.). Central nervous system stem cells also proliferate in response to the EGF stimulus (Reynolds & Weiss).
Primary Neuronal Culture: Standardized Media and Reagents
BrainPhys™ Neuronal Medium for Improved Neuronal Function
Cytokines, Chemokines and Growth Factors
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(A) The biological activity of Human Recombinant EGF was tested by its ability to promote the proliferation of BALB/c 3T3 cells. Cell
proliferation was measured using a fluorometric assay method. The EC50 is defined as the effective concentration of the growth factor at
which cell proliferation is at 50% of maximum. The EC50 in the above example is 0.1 ng/mL.
(B) 2 μg of Human Recombinant EGF was resolved with SDS-PAGE under reducing (+) and non-reducing (-) conditions and visualized by
Coomassie Blue staining. Human Recombinant EGF has a predicted molecular mass of 6.2 kDa.
STEM CELLS Translational Medicine 2016 NOV
A Novel Protocol for Directed Differentiation of C9orf72-Associated Human Induced Pluripotent Stem Cells Into Contractile Skeletal Myotubes
Swartz EW et al.
: Induced pluripotent stem cells (iPSCs) offer an unlimited resource of cells to be used for the study of underlying molecular biology of disease, therapeutic drug screening, and transplant-based regenerative medicine. However, methods for the directed differentiation of skeletal muscle for these purposes remain scarce and incomplete. Here, we present a novel, small molecule-based protocol for the generation of multinucleated skeletal myotubes using eight independent iPSC lines. Through combinatorial inhibition of phosphoinositide 3-kinase (PI3K) and glycogen synthase kinase 3β (GSK3β) with addition of bone morphogenic protein 4 (BMP4) and fibroblast growth factor 2 (FGF2), we report up to 64% conversion of iPSCs into the myogenic program by day 36 as indicated by MYOG+ cell populations. These cells began to exhibit spontaneous contractions as early as 34 days in vitro in the presence of a serum-free medium formulation. We used this protocol to obtain iPSC-derived muscle cells from frontotemporal dementia (FTD) patients harboring C9orf72 hexanucleotide repeat expansions (rGGGGCC), sporadic FTD, and unaffected controls. iPSCs derived from rGGGGCC carriers contained RNA foci but did not vary in differentiation efficiency when compared to unaffected controls nor display mislocalized TDP-43 after as many as 120 days in vitro. This study presents a rapid, efficient, and transgene-free method for generating multinucleated skeletal myotubes from iPSCs and a resource for further modeling the role of skeletal muscle in amyotrophic lateral sclerosis and other motor neuron diseases. SIGNIFICANCE Protocols to produce skeletal myotubes for disease modeling or therapy are scarce and incomplete. The present study efficiently generates functional skeletal myotubes from human induced pluripotent stem cells using a small molecule-based approach. Using this strategy, terminal myogenic induction of up to 64% in 36 days and spontaneously contractile myotubes within 34 days were achieved. Myotubes derived from patients carrying the C9orf72 repeat expansion show no change in differentiation efficiency and normal TDP-43 localization after as many as 120 days in vitro when compared to unaffected controls. This study provides an efficient, novel protocol for the generation of skeletal myotubes from human induced pluripotent stem cells that may serve as a valuable tool in drug discovery and modeling of musculoskeletal and neuromuscular diseases.
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