Hossein Faghih - Stem Cells and Regenerative Medicine, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
Arash Javeri - Stem Cells and Regenerative Medicine, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
Masoumeh Fakhr Taha - Stem Cells and Regenerative Medicine, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran

Background and Aim: Dopaminergic (DA) neurons regulate locomotionand emotion. Degeneration of DA neurons is involved in differentneurodegenerative disorders including Parkinson’s disease (PD). Cellreplacement therapy has been regarded as a promising strategy for thetreatment of PD. Human DA neurons have been generated from multipletypes of stem cell in vitro. We aimed to study the efficiency of generatingdopaminergic neurons from ADSCs under low-serum and serum-freeculture conditions. Methods: Human adipose tissue samples were collected byabdominoplasty and ADSCs were isolated by collagenase treatment.For dopaminergic differentiation, third to fifth-passaged ADSCs wereinduced by a dopaminergic cocktail containing SHH, bFGF, FGF8,B27, and BDNF in serum-free (Neurobasal medium) or low-serum(DMEM+1% FBS) condition for 12 days. The expression of severalneuronal and dopaminergic markers was compared by RT-PCR, qPCR,immunocytochemistry and flow cytometry analyses. Also, secretion ofdopamine was measured by high-performance liquid chromatography(HPLC).Results: RT-PCR results showed that NSE, NEFL, NURR1, EN1, GLI1,PTX3, TH, VMAT2 and GIRK2 mRNAs were expressed in both serum-freeand low-serum conditions. Based on the qPCR analysis, the expression ofNEFL and NURR1 was not different between two protocols, while NSE,EN1, GLI1 and TH expression in serum-free condition was significantlyhigher than the low-serum condition. A significant proportion ofdifferentiated cells were positively immunostained for TH and MAP2proteins. Flow cytometry analysis revealed that TH protein was expressedin about 41% and 13% of the cells in the serum-free and low-serumconditions, respectively. Moreover, the cells differentiated under serumfreecondition released a higher amount of dopamine in response to KClinduceddepolarization.Conclusion: The results of the current study indicated that human ADSCscan be induced by a growth factor cocktail to produce dopaminesecretingcells. Dopaminergic differentiation in serum-free (neurobasalmedium) condition was more effective than low-serum (DMEM+1% FBS)condition. Therefore, the serum-free condition may be used to producedopaminergic cells for the future cell replacement therapy in Parkinson’sdisease.

ADSC; Differentiation; Dopaminergic neuron; Serum-free; Low-serum