The Impact of Mir-302/367 Cluster on Pluripotency and Dopaminergic Differentiation of Human Adipose Tis-sue-Derived Stem Cells

Background: Adipose tissue-derived stem cells (ADSCs) se-crete angiogenic, anti-inflammatory, immunomodulatory and neuroprotective factors. Also, several studies have documented neural and dopaminergic differentiation of the ADSCs which raises the hope of using these cells for the treatment of neuro-degenerative disorders including Parkinson s disease. However, for clinical application, highly efficient techniques are required to generate a purified population of dopaminergic neurons. One solution may be reprogramming of the ADSCs towards a more pluripotent state by different strategies. Here, we used miR-302/367 cluster to improve pluripotency and dopaminergic dif-ferentiation potential of human ADSCs.Materials and Methods: Third-passaged ADSCs were trans-fected with the pTD.CMV-hsa-miR-302bcad/367-EF1-GFP-T2A-Puro (System Biosciences, USA) using a Neon Transfec-tion system (Invitrogen, Thermo Fisher Scientific, USA). After three weeks, the ADSCs were assessed for the expression of pluripotency genes. Also, dopaminergic differentiation of the control and transfected ADSCs was induced by a growth fac-tor cocktail consisting of SHH, FGF-8, b-FGF and BDNF and analysed by RT-PCR, quantitative real-time PCR, immunocyto-chemistry and western blot.Results: Transfection of the ADSCs with miR-302/367 cluster upregulated the expression of OCT4, NANOG, SOX2, REX1 and NODAL mRNAs. Both the control and miR-302/367-transfected cells were successfully differentiated into dopamin-ergic neurons and expressed some genes associated with dopa-minergic differentiation, including EN1, GLI1, NURR1, TH, and GIRK2 mRNAs and TUJ1 and TH proteins while TH and GIRK2 expressions were upregulated.Conclusion: These findings show that miR-302/367 cluster re-programs human ADSCs towards a more pluripotent state and improves dopaminergic differentiation potential of the ADSCs which may be in favour of future clinical application of these cells.

Keywords:

ADSC , miR302/367 , Dopaminergic Differentiation , Pluripotency