丸野内　棣、鳥羽　慎也、大熊　真人、秋山　秀彦、丸野内　暢彦 (藤田保健衛生大学　総合医科学研究所　応用細胞学研究部門)

Recently bone marrow derived stem cells (MSCs) have been shown to yield a multitude of cell types including neuronal cells. MSCs have advantages to be easily collected and returned to the host after some treatments compared to ES cells. We tried to establish the culture condition in which MSCs were differentiated into neuronal cells at a high frequency and were maintained for a long time as the differentiated state.

Almost all of MSCs were induced to neuronal cells by the method of Woodbury et al and maintained as long as two to three weeks in vitro under the condition established by us. We studied changes in gene expression during the differentiation of MSCs into neuronal cells by immunostaining method using antibodies against specific neuronal markers. As a result, antibodies for neurofilament M, MAP2 and neuron specific enolase stained induced cells. Moreover, an antibody directed against serotonin stained a number of differentiated cells.

Next, we prepared total RNA from induced cells and analyzed the expression of neuronal markers by RT-PCR method. The amount of transcripts of Nurr1, NeuroD1, neurofilament M and H, MAP2, neuron specific enolase, and tryptophan hydroxylase increased after induction. These results suggested that that MSCs differentiated into serotonergic neuron-like cells and maintained for a long time under the condition modified by us.

Finally we examined electrophysiological characteristics of differentiated cells and detected voltage-gated Na+ channels, which was sensitive to tetrodotoxin.