Survival, neuron-like differentiation and functionality of mesenchymal stem cells in neurotoxic environment: the critical role of erythropoietin.

Mesenchymal stem cells (MSCs) can ameliorate symptoms in several neurodegenerative diseases. However, the toxic environment of a degenerating central nervous system (CNS) characterized by hypoxia, glutamate (Glu) excess and amyloid beta (Abeta) pathology may hamper the survival and regenerative/replacing capacities of engrafted stem cells. Indeed, human MSC (hMSC) exposed to hypoxia were disabled in (i) the capacity of their muscarinic receptors (mAChRs) to respond to acetylcholine (ACh) with a transient increase in intracellular [Ca(2+)], (ii) their capacity to metabolize Glu, reflected by a strong decrease in glutamine synthetase activity, and (iii) their survival on exposure to Glu. Cocultivation of MSC with PC12 cells expressing the amyloid precursor protein gene (APPsw-PC12) increased the release of IL-6 from MSC. HMSC exposed to erythropoietin (EPO) showed a cholinergic neuron-like phenotype reflected by increased cellular levels of choline acetyltransferase, ACh and mAChR. All their functional deficits observed under hypoxia, Glu exposure and APPsw-PC12 cocultivation were reversed by the application of EPO, which increased the expression of Wnt3a. EPO also enhanced the metabolism of Abeta in MSC by increasing their neprilysin content. Our data show that cholinergic neuron-like differentiation of MSC, their functionality and resistance to a neurotoxic environment is regulated and can be improved by EPO, highlighting its potential for optimizing cellular therapies of the CNS.

Use of solid-phase extraction to determine ergosterol concentrations in plant tissue colonized by fungi.

At present, the ergosterol and acetate-to-ergosterol techniques are generally considered to be the methods of choice to quantify fungal biomass, growth rate, and productivity under natural conditions. Both methods rely on the accurate isolation and quantification of ergosterol, a major membrane component of eumycotic fungi. Taking advantage of the solid-phase extraction (SPE) technique, we present a novel method to determine the ergosterol concentration in lipid extracts derived from plant tissues and dead organic matter colonized by fungi. In this method, a primary alkaline extract is acidified and passed through a reversed-phase (C(inf18)) SPE column. The column is then washed with an alkaline methanol-water solution to eliminate interfering substances and increase pH and is thoroughly dried in air. Ergosterol is eluted with alkaline isopropanol. This eluting solvent was chosen to produce a strongly basic pH of the final extract and thus confer stability on the ergosterol molecule before high-performance liquid chromatography analysis. The recovery of ergosterol from plant tissues and the O(infhf) horizon of a woodland soil ranged from 85 to 98%, and the overall extraction efficiency was similar to that obtained by a conventional procedure involving liquid-liquid extraction. Potential pitfalls of ergosterol analysis by SPE include (i) insufficient acidification before sample loading on the extraction column, resulting in a poor affinity of ergosterol for the sorbent; (ii) incomplete drying of the sorbent bed before the elution step; and (iii) chemical breakdown of ergosterol after elution, which was found to be related to a low pH of the final extract and a high concentration of matrix compounds. When these pitfalls are avoided, SPE is an attractive alternative to existing methods of ergosterol analysis of environmental samples.