Differentiation Potency of New Mesenchymal Populations for Neural Cell Lineage

Objectives: Bone marrow stromal cells (BMSCs) have been shown to be a heterogeneous population of mesenchymal stem cells (MSCs) which is important in tissue regeneration including periodontal tissues and dentin-pulp complex. However, there was not any established method to isolate MSCs. Recently, we have established a new method to purify bone marrow-derived populations showing a high frequency of osteoprogenitors and named them highly purified osteoprogenitors (HipOPs). Although our previous studies demonstrated that HipOPs are enriched with MSCs and have a high differentiation potential into osteoblasts, adipocytes and chondrocytes compared to BMSCs, their differentiation potential for neural cell lineage has not been clarified. In this study, we evaluated the efficacy of HipOPs as a resource for neural cells.
Methods: BMSCs were isolated from C57BL/6J mice and purified through negative selection using magnetic beads technique to obtain HipOPs. Cells were cultured in the presence of basic fibroblast growth factor and epidermal growth factor. When mature neurospheres were observed, they were separated and dissociated into single-cell suspension and cultured under the same conditions as mentioned above. Neurospheres were also applied to immunostaining with nestin, which is expressed on neural cells. To evaluate the frequency of neural progenitors in BMSCs and HipOPs, we practiced a limiting dilution assay.
Results: HipOPs generated neurospheres, which is typical for neural stem cells. After passage, they generated secondary and tertiary neurospheres, thus we confirmed that HipOPs have the self-renewal potency, indicating that they are stem cells. Immunocytochemical analysis showed that these spheres were nestin positive. According to the limiting dilution assay, the neural stem cell frequency of HipOPs was 1/402, whereas that of BMSCs was 1/48990 (t=36.91, p<0.001).
Conclusions: These results demonstrated that HipOPs have a high differentiation potential for neural cell lineage.