Enrichment of Motor Neuron-like Cell Precursors Derived from Mouse Embryonic Stem Cells by Density Gradient Centrifugation

Abstract

Abstract:Objective:Our objective was to isolate motor neuron-like cell precursors (MNLCPs) derived from mouse embryonic stem cells (mESCs) within embryoid bodies (EBs) for the development of drug screening assays and transplantation therapies in motor neuron disorders. EBs, induced with native Shh protein (or agonists of Shh pathway) and retinoic acid, contain variable proportions of MNLCPs and undifferentiated cells. Undifferentiated cells may interfere in drug screening assays or proliferate following transplantation if not sufficiently removed from the culture. We have developed a method based on density gradient centrifugation to enrich MNLCPs.Methods:mESCs (HBG3:eGFP: HB9) were expanded and differentiated using a modified protocol by Wichterleet al.(2008). EBs containing GFP (+) MNLCPs and undifferentiated cells were gently dissociated into single cells by chemical and enzymatic treatments without trituration. MNLCPs were recovered after centrifugation of dissociated cells on Optiprep^TM8%~20% step-gradient. The amount of GFP (+) MNLCPs was determined by flow cytometry.Results:Our results show that mESCs grown on gelatinized plates prior to EB formation, decreases the ability of mESCs to differentiate into MNLCPs. mESCs were grown on gelatin, gelatin with PMEFs, and PMEFs alone and found that growing mESCs on PMEF yielded GFP (+) MNLCPs at 54.1% ± 11.0% ( ±s；n=12) compared with gelatin 2.8% ±1.1% ( ±s；n=9). We obtained enriched fractions containing on average 87.7% ± 5.5%( ±s；n=3) GFP(+) MNLCPs by density gradient centrifugation.Conclusion:Our data support that dissociation without trituration and density gradient centrifugation can be used to significantly enrich MNLCPs retaining high viability without the use of a cell sorter. Further characterization of MNLCPs is necessary to design an appropriate assay such as physiological relevancein vitroandin vivoand phenotype such as neurite outgrowth, and the ability to form neuromuscular junctions.

Key words:embryonic stem cells,density gradient,motor neuron

Stephanie Richardson Milazi Ph.D., Benjamin Rix Brooks M.D., Jean-Luc Mougeot Ph.D. . Enrichment of Motor Neuron-like Cell Precursors Derived from Mouse Embryonic Stem Cells by Density Gradient Centrifugation[J]. Acta Neuropharmacologica, 2011, 1(1): 16-26.

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