Conditioned medium of olfactory ensheathing or neuroblastoma B104 cells promotes differentiation of human mesenchymal stem cells from adipose tissue toward a neural phenotype

The mammalian olfactory system is one of the few areas of thecentral nervous system able of continuous neurogenesis throughoutlifetime supported by peculiar glial cells of the olfactory nerve,called Olfactory Ensheathing Cells (OECs). They share phenotypiccharacteristics with Schwann cells (SCs) and astrocytes and representa source of several trophic factors. On the other hand, B104neuroblastoma cells are recognized to induce differentiation ofneural stem cells into oligodendrocyte precursor cells. Consideringas described, in this work, we studied the effect of both B104 andOECs conditioned medium on cultured adipose tissue-derived mesenchymalstem cells (AT-MSCs) and verified if these conditionedmedia were capable of inducing them to a neuronal phenotype. Inorder to this goal, AT-MSCs cultures were obtained from 10donors undergoing abdominal liposuction procedures. OECs wereisolated from 2-day old rat pup (P2) olfactory bulbs, the mediumwas collected after 24-48 h and stored (-20°C) until further use.The B104 neuroblastoma cell line was maintained in DMEM/FBS,after 3 days the medium was collected, filtered and stored at -80°C. After reaching confluence, AT-MSCs were trypsinized andsubcultured in multi-well plates for 24 h. The medium was removedand replaced with OECs-CM and/or B104-CM. Some wells wereused as control and grown only with DMEM/FBS. Some sampleswere used for immunocytochemistry and others for flow cytometry.Some neural markers, such as nestin, protein gene product 9.5(PGP 9.5), microtubule-associated protein 2 (MAP2), glial fibrillaryacidic protein (GFAP), and neuron cell surface antigen(A2B5) were examined 24 h and 7 days after the treatment in controland conditioned media-treated cultures. The results show thatAT-MSCs treated with either media express markers of progenitorand mature neurons (nestin, PGP 9.5 and MAP2) in a timedependentmanner. They display morphological features resemblingneuronal cells, and result negative for GFAP and A2B5, astrocyteand oligodendrocyte markers, respectively. This study demonstratesthat AT-MSCs could be influenced by the environment toward aneuronal phenotype. This culture system may offer many advantagesas potential material for replacement therapy in centralnervous system degenerative diseases.