Experimental study of cultivation and identification of rat bone marrow mesenchymal stem cells and their differentiation into neural stem cells

Abstract:

Objective：To culture in vitro and identify rat bone marrow mesenchymal stem cells（BMSCs），to study their biological char－acteristics and to tentatively explore the feasibility of differentiation of BMSCs into neural stem cells（NSCs） induced by acidic fibrob－last growth factor（aFGF）. Methods：Rat BMSCs were isolated and cultured by the whole bone marrow adherent culture method. Mor－phological characteristics of BMSCs were observed under the inverted phase contrast microscope and cell growth curves were drawn by MTT method. Cell cycle and surface markers were detected with flow cytometry（FCM）. Rat BMSCs were induced to differentiate into NSCs by 80 ng/ml aFGF of DMEM/F12 medium. Cell morphological changes were continuously observed after using aFGF. Im－munofluorescence staining was used to detect the expressions of Nestin and neuron specific enolase（NSE）. Results：Rat BMSCs showed adherent growth and was spindle shaped or in polygonal appearance. Growth curves of the 1st，3rd，5th-generation BMSCs were in S shape and their activities were not significantly different. FCM showed that 94.34% BMSCs were in G0/G1 phase of the cell cycle，having strong proliferation ability. FCM detected that CD29，CD54 and CD90 were positively expressed，but CD45 was negative－ly expressed. At 6 h after the induction，cell bodies became to shrink into oval or sphere，and stretched out long and thin protrusions. Immunofluorescence staining showed that Nestin was positively expressed. Bipolar and multipolar cells were constantly increased after continuous induction. At 6 d after the induction，cells interconnected into a mesh and became typical neuron-like cells. NSE was posi－tively expressed. Conclusions：Whole bone marrow adherent culture method can cultivate highly purified rat BMSCs with stem cells’ characteristics：growing stably，proliferating rapidly and be multiple-passaged. By the induction of aFGF，BMSCs have the potentiality of differentiating into NSCs，which can further differentiate into neuron-like cells.