[Show abstract][Hide abstract]ABSTRACT: Fibroblast-derived dopaminergic neurons. (A) The ALN polycistronic vector is effective in converting fibroblasts to dopaminergic neurons, demonstrated by immunocytochemistry for the neuronal marker TUJ1 (14.9±2.3% positive) and the dopaminergic marker tyrosine hydroxylase (9.1±0.9% positive); the pan-nuclear marker DAPI is also shown. Scale bars 50 µm. (B) Action potential firing characteristics of fibroblast-derived dopaminergic neurons. Four overlapping traces are depicted derived from whole-cell current clamp recording of a representative induced dopaminergic neuron, elicited in response to hyperpolarizing and depolarizing current injection, increased from −20 to +10 pA in 10 pA increments. (C) Voltage-dependent sodium currents in fibroblast-derived dopaminergic neurons. Membrane potential was initially held at −90 mV and incrementally increased from −60 to +40 mV in 5 mV depolarizing steps. (D) Spontaneous action potential firing, consistent with a dopaminergic neuron pacemaker phenotype. The recording was conducted at resting membrane potential (−57 mV).
[Show abstract][Hide abstract]ABSTRACT: Direct cellular reprogramming is a powerful new tool for regenerative medicine. In efforts to understand and treat Parkinson's Disease (PD), which is marked by the degeneration of dopaminergic neurons in the midbrain, direct reprogramming provides a valuable new source of these cells. Astrocytes, the most plentiful cells in the central nervous system, are an ideal starting population for the direct generation of dopaminergic neurons. In addition to their potential utility in cell replacement therapies for PD or in modeling the disease in vitro, astrocyte-derived dopaminergic neurons offer the prospect of direct in vivo reprogramming within the brain. As a first step toward this goal, we report the reprogramming of astrocytes to dopaminergic neurons using three transcription factors – ASCL1, LMX1B, and NURR1 – delivered in a single polycistronic lentiviral vector. The process is efficient, with 18.261.5% of cells expressing markers of dopaminergic neurons after two weeks. The neurons exhibit expression profiles and electrophysiological characteristics consistent with midbrain dopaminergic neurons, notably including spontaneous pacemaking activity, stimulated release of dopamine, and calcium oscillations. The present study is the first demonstration that a single vector can mediate reprogramming to dopaminergic neurons, and indicates that astrocytes are an ideal starting population for the direct generation of dopaminergic neurons.
[Show abstract][Hide abstract]ABSTRACT: Calcium oscillations in an astrocyte-derived dopaminergic neuron. The genetically-encoded calcium indicator GCaMP3 is expressed under the control of the neuron-specific MAP2 promoter. Fluorescence level increases with increased levels of intracellular calcium. Rhythmic oscillations of calcium levels are seen, consistent with the midbrain dopaminergic phenotype.