Isolation and culture of adipose-derived stromal cells and a two-step protocol to induce their differentiation into neural cells

Objective To explore a new method for in vitro culture and differentiation induction of rat adipose-derived stromal cells(ADSCs)into neural cells.Methods The retroperitoneal adipose tissue of adult SD rats was carefully dissected and digested using type Ⅰ collagenase.After centrifugation.the stromal cell pellet was resuspended in DMEM/F12 containing 10%fetal bovine serum (FBS).The ADSCs in passage 5 were harvested and cultured in serum-free Neurobasal (NB)medium supplemented with 20 ng/mL basic fibroblast growth factor(bFGF),20 ng/mL of epidermal growth factor (EGF)and N2(1:100)to induce neurosphere formation.The neurospheres in passage 2 were incubated in NB medium supplemented with 0.5 μmol/L all-trails-retinoic acid(RA),1%FBS,5%horse serum,and 50 ng/mL of brain-derived neurotrophic factor (BDNF)to induce the their differentiation into neuron-and glial-like cells.Immunofluorescent staining was performed to identify the differentiated cells. Results Early after inoculation,the spherical ADSCs were suspended in the medium and began to adhere to the wall of the culture flask 24 h after the inoculation.With the increase of the cell density in the cell culture,a fibroblast-like cell monolayer occurred.Neurosphere formation was observed 5-7 days after culturing the ADSCsin serum-free NB medium,and the neurospheres rapidly proliferated and reached a diameter of 100μm at 14 days of culture.The neurospheres further terminally differentiated into neuron-and glial-like cells with spherical morphology and clearly visible cell nuclei.The differentiated cells extended long and thin cell processes or reticular cell processes,and numerous cells established networks through the cell processes.Immunofluorescent staining showed that the neurospheres were positive for nestin (with a positivity rate of 75.62%±1.34%),and 57.62%±4.92%of the terminally differentiated cells expressed β-tubulin Ⅲ,a marker of immature neurons,and 11.25%±3.87%were positive for MAP2ab,a marker of mature neurons.Some of the differentiated cells were positive for glial fibrillary acidic protein (GFAP)expression(18.34%±3.87%)and galactoeerebroside(GalC)(14.35%±3.98%). Conclusions ADSCs from adult rat adipose tissue Can produce a large quantity of stable multipotent daughter cells.The two-step method for inducing the differentiation of ADSCs Can yield high rates of nestin-and β-tubulin Ⅲ-positive cells and some MAP2ab-positive cells. Key words: Adipose-derived stromal cells; Induction; Cell differentiation; Neuron-like cells