Elevation of cAMP in Mesenchymal Stem Cells Transiently Upregulates Neural Markers Rather than Inducing Neural Differentiation

Abstract

The aims of this research were to examine the neural expression profile of undifferentiated mesenchymal stem cells (MSCs), to define a serum-free environment that would support the survival of MSCs, and to assess the effects of elevated cyclic adenosine monophosphate (cAMP) levels on MSC morphology and expression of neural markers. The neural profile of MSCs was assessed using immunocytochemistry and real-time polymerase chain reaction (PCR) techniques. These MSCs were then cultured in varying serum-free environments, with flow cytometry analysis of cell viability and apoptosis. The effects of forskolin and 8 bromo-cAMP treatment on MSC morphology and expression of neural markers were assessed using light microscopy, immunocytochemistry and real-time PCR analysis. Expression of the neural markers nestin, vimentin, beta III tubulin, glial fibrillary acidic protein, and the tropomyosin-related kinases was demonstrated in normal undifferentiated MSCs. Furthermore, MSCs cultured in serum-free conditions containing ascorbic acid 2-phosphate demonstrated greater long-term survival and reduced apoptosis. In contrast, forskolin increased the percentage of cells undergoing apoptosis. Culture in the presence of forskolin induced a 6-fold increase in beta III tubulin expression after a 6-h exposure time, which was accompanied by dramatic changes in morphology. However, this effect was transient, with the cells reverting to their normal morphology by 24 h. Treatment with 8 bromo-cAMP induced similar increases in beta III tubulin expression without such dramatic morphological changes. Factors which increase the concentration of cAMP induce transient changes in expression of neural markers that appear to be the result of cellular adaptation to changes in culture conditions rather than a real process of neural differentiation.