You are hereDecember 28, 2014 | ESCs/iPSCs
Quick and Easy Recipe for Mature Neurons from iPSCs
Review of “Efficient and Cost-Effective Generation of Mature Neurons from Human-Induced Pluripotent Cells” from Stem Cells TM by Stuart P. Atkinson
Induced pluripotent stem cell (iPSC) technology holds great promise; not only for the generation of patient specific stem cells and their useful derivatives, but also for the modeling of various diseases. However, efficient and cost effective protocols are required to allow this technology to flourish. Understanding this, the group of Frédérique Magdinier (INSERM, Marseille, France) now describe their recently developed simple method for the differentiation of iPSCs into neurons .
This technique is displayed graphically in the adjoined figure, and will be explored herein. The authors first plated clumps of hiPSC colonies on a Matrigel growth substrate and added Neurobasal medium (NB), bFGF and EGF (Differentiation Medium + [DM+]) alongside 2% DMSO. After 16 hours, the researchers replaced the medium with DM+ and expanded the cells for 15 days. During this process, cells lost their pluripotent identity, gaining a flat spindle-like neuroepithelial morphology and the expression of the NSC marker gene NESTIN from day 4 onwards. At this stage, the group could culture NSCs as cell clumps on fibronectin long term with little loss of the expression the NSC marker genes NESTIN, PAX6 and SOX1. NSCs could also be frozen at this time and thawed without loss of these characteristics. Overall, the authors demonstrated this to be a highly reproducible strategy with similar yields obtained from different iPSCs.
For further differentiation, the group mechanically separated cells using a 23-gauge needle and plated them onto laminin with DM without bFGF and EGF which generated III tubulin- and NeuN-positive neurons after 5-7 days. Assessment of neurons maintained on laminin for up to 35 days demonstrated the hoped for membrane properties; the vast majority of cells were able to generate and propagate action potentials, and cells expressed receptor-operated channels (82% of cells replied to GABA, 71% responded to glycine, and 25% responded to acetylcholine). These cells also demonstrated excellent further differentiation propensities, as the further application of FGF8 and SHH for 48 hours (See Figure) mediated the differentiation of neural cultures into a 80% pure culture of TH+ dopaminergic neurons.
This simple differentiation schemes uses no feeders, does not require embryoid body, rosette or neurosphere formation, or cell sorting, and is cheaper due to the absence of several widely used factors (BDNF, Noggin, NT3, or GDNF). Importantly the protocol also yields large quantities of neural stem/progenitor cells. This quick, simple and cheap differentiation protocol therefore has the potential to further expand iPSC-based disease modeling, and to create the vast amounts of cells required for transplantation.
- Badja C, Maleeva G, El-Yazidi C, et al. Efficient and cost-effective generation of mature neurons from human induced pluripotent stem cells. Stem Cells Translational Medicine 2014;3:1467-1472.