The Thai Journal of Veterinary Medicine


Mouse embryonic stem (ES) cells have been served as potential model for investigation of underlying mechanisms at cellular and molecular levels of neurological disorders. The improvement of motor neuron differentiation is prospected to gain more understanding aimed at overcoming several incurable motor neuron diseases. In this study, we examined the effects of selective inhibitor of TGF-β type I receptor on the efficacy of neuronal differentiation of mouse embryonic stem cells toward motor neuron. Pluripotent ES cells were induced to form as EB in suspension medium supplemented with retinoic acid using -4/+4 protocol. Thereafter, 8-day old EBs were further induced to differentiate into motor neurons on monolayer culture. Our result demonstrated that the aggregated ES cells differentiated into neuronal progenitor cells and neurons as examined with Pax-6 and Tuj-1. Quantitative RT-PCR analysis revealed that treatment of the EBs with selective TGF-β1 inhibitor up-regulated the motor neuron progenitor (Olig2) at higher levels than that obtained from the control (4.20±0.20 vs. 0.73±0.09, p < 0.01). In contrast, mRNA expression levels of motor neuron (Hoxc8) of the control group were significantly higher than the TGF-β1 inhibitor treated group (14.73±2.6 vs. 2.37±0.42, p < 0.01). Immunocytochemistry demonstrated that the differentiated cells expressed a neuronal marker (Tuj-1), motor neuron progenitor marker (Olig2) and developing motor neuron progenitor (Isl-1), all of which are essential for generation of spinal motor neurons during neural tube formation. Furthermore, a small proportion of differentiated cells were also positive for choline acetyltransferase (ChAT) a marker for functional motor neurons. We concluded that modifying TGF-β signaling affected the generation and differentiation fate of motor neuron progenitor cells.

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