Analysis of K+ channels in the maturation of dopaminergic neurons

Abstract

When induced sufficiently in vitro, mesenchymal stem cells (MSCs) may undergo reprogramming toward the neuroectodermal lineage. This study attempted to derive mature, functional dopaminergic (DA) neurons from primary MSCs, in pursuit of neuroregenerative therapy targeting Parkinson’s disease (PD), a chronic disorder designated by progressive DA neurodegeneration. The efficacy of the employed induction protocol in successfully deriving DA neurons was evaluated by morphological, transcriptomic, and proteomic analysis of cells as they underwent treatment. Morphological assessment revealed that, in response to treatment, MSCs ceded their typical fibroblastic morphology to adopt a characteristic mature neuronal cell morphology. Transcriptomic RT-PCR (real-time polymerase chain reaction) analysis, moreover, indicated that early phases of treatment were indeed successful at provoking MSC lineage reprogramming. This was substantiated by the observed downregulation of typical MSC markers (CD90) and the concomitant upregulation of neuroprogenitor markers (Sex determining region Y-box 2 and Nestin). Later phases of treatment subsequently upregulated multiple mature neuronal markers (neuronal nuclear protein, Kv1.1, Microtubule-associated protein 2, SK1, and SK3) and DA-specific markers (Tyrosine hydroxylase). These treatment-induced mRNA expression patterns were largely corroborated at protein level by Western blotting, and strongly signified efficacy of the adopted induction protocol in reprogramming MSCs, specifying the DA neuronal cell fate, and provoking terminal differentiation. The successful derivation of mature, functional DA neurons from stem cells not only contributes to translational efforts pursuing large-scale clinical administration of stem cell-based therapy, but also enables the generation of cellular models of PD in the laboratory, which hold tremendous potential as research tools to elucidate the obscure cellular mechanisms underlying PD pathogenesis.