Induced Differentiation To Motor Neurons From Neural Stem Cells Of Astrocytic Origin In The Rat

Destruction of the motor neurons will lead to loss of innervation of the somatic muscle, which has long been considered an illness with no remedy. The only possible treatment is to substitute the injured motor neurons by neurons differentiated from stem cells. It has recently been reported that embryonic stem cells can be induced to differentiate to motor neurons. However, the use of embryonic stem cells has innate problems, such as immune rejection and tumor formation. The ideal source of motor neurons should be the cells from the patients themselves which has the potential to be induced to motor neurons. Our previous study demonstrated that mature astrocyte has the potential of de-differentiation to neural stem cell. The present study was aimed to investigate if the neural stem cells of astrocytic origin can be induced to motor neurons.Astrocytes derived from 15-20 day postnatal rat spinal cord were cultured in modified chemically defined medium (MCM). Immunostaining,Western blot,Fluorescence Activated Cell Sorting,Transfection and Time-lapse technologies were used to investigate the de-differentiation of astrocytes in vitro. To test the identity of the neural stem cells, neurospheres culture condition was established in the presence of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Retinoic acid (RA) and sonic hedgehog (Shh) inducing factors were used for the differentiation of motor neurons.Here, we show that astrocytes cultured in the defined culture system can de-differentiate and grow neurospheres in the presence of EGF and bFGF. The cells from the neurospheres were successfully passaged once per week to at least 16 passages. After the neurosphere cells were plated at a density of 100 cells/well in 48 wells of the 96-well plates, 22.7±9.9 new neurospheres were formed in each well after 3 weeks, with sizes were from 50-150μm. When 1×10~6 neurosphere cells were plated, (6.6±0.4)×10~6 cells were obtained in 7 days. These neurospheres express Nestin, a neural stem/progenitor cell marker, have the ability of self-renewing and differentiate into neurons and glia-lineage cells. These data indicate their neural stem cell identity. Furthermore, the neurospheres were induced by RA and Shh factors. After 5days induction, we found that 97.7 %±0.5 % cells are olig2+ spinal motor neuron progenitors, and 97.6 %±0.5 % cells areβIII-tubulin+. Almost all of olig2+ cells areβIII-tubulin+. For additional 2 weeks mature differentiation, we observed that 24.4 %±1.1 % cells are HB9+ spinal motor neurons and all of these cells areβIII-tubulin + and ChAT+. Whereas, the cells without RA and Shh treatment generated significantly fewer HB9-ir motor neurons. These data demonstrated that RA and Shh can significantly stimulate the neural stem cells to produce motor neurons.The results demonstrated that neural stem cells of astrocytic origin could be induced to differentiate into motor neurons and their progenitor cells with rich harvest. Furthermore, it has been reported that astrocytes can be readily obtained via biopsy from the cerebral cortex of the patient, rendering autologous transplantation possible.