(96c) cAMP Induced Independent Early-Phase Neuron-Like Morphological Change and Late-Phase Neural Differentiation Behavior in Rat Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) were shown to undergo neural differentiation upon stimulation with cAMP increasing agents, based on both the appearance of neural-like morphology and expression of neural markers. However, the neural-like morphology occurred within one hour, which is too quick for real differentiation, casting doubt on the ability of MSCs to differentiate into neural lineage cells by cAMP increasing agents. As cAMP increasing agents are widely used in differentiation cocktails, an understanding of how cAMP causes morphological changes and whether such morphological changes are real indicatiors of differentiation is important for future design of neural differentiation in MSCs. Therefore, we specifically evaluated the effect of cAMP on MSCs morphological changes. Our result indicated that cAMP induced neural-like morphology is the result of retraction of the cell body towards the center rather than neurite outgrowth and is a transcriptionally and translationally independent process. Reorganization of cytoskeleton occurred within 10 minutes after stimulation. Star-like and spindle-like cells are the most abundant subpopulations in MSCs and were affected the most by cAMP stimulation. However, as the treatment time increased, they either reverted back to their original shape or lifted up. Very few cells were able to retain their morphological changes. Measurement of cAMP levels showed that the intracellular, correspondingly, cAMP peaked within one hour and then gradually decreased. Re-stimulation with cAMP increasing agents did not further induce the neural-like morphological changes or intracellular cAMP peak. The latter is possibly due to the desensitization of adenylyl cyclase (AC), the enzyme that produces cAMP. The classical cAMP signaling component protein kinase A (PKAc), which is involved in regulating the neural-like morphological changes in MSCs, decreased continuously with cAMP stimulation, which may explain the decrease in the neural-like morphology over time. However, it is unclear which factor or factors downstream of PKA are involved in regulating the morphological changes. Possible candidates include the Rho family proteins. Work is underway, both experimentally and computationally, to elucidate the pathways involved in the regulation of cAMP-induced neural-like morphological changes.