Human astrocytes were converted into neurons by the miR-302/367 cluster in vitro, without pre-treatment with valproic acid (VPA).

<p><b>(A)</b> Human induced neurons expressed neuroblast marker [doublecortin (DCX)] and neuronal markers (TUJ1 and NeuN) at 8 and 10 days post in vitro (DPI) when they received the miR-302/367 cluster. <b>(B)</b> Quantification of immunostaining data provided in A. Scale bar: 50 μm.</p

Conversion of astrocytes into neuronal cells which did not pass through the pluripotent stage.

<p><b>(A)</b> Expressions of Oct4 and Nanog genes as pluripotent markers were not detected following miR-302/367+ valproic acid (VPA) treatment. n = 3. Scale bar: 50 μm. (B) The brain tissue of animals treated with the miR-302/367 cluster were investigated for the teratoma formation. No teratoma formation was observed at 60 days post-injection (dpi). n = 2. Scale bar: 50 μm.</p

A number of cells transduced with the miR-302/367+GFP cluster following valproic acid (VPA) pre-treatment showed neuronal fate as determined by immunostaining against NeuN.

<p><b>(A)</b> Animals received miR-302/367+GFP expressing vectors. VPA showed GFP⁺ cells that expressed NeuN. <b>(B)</b> Quantification of immunostaining data showed that GFP⁺/NeuN⁺ cells were produced in the miR-302/367+VPA group, particularly at 14 days post-injection (dpi). ***p<0.001 compared to 7 dpi and ⁺⁺p<0.01 compared to miR-302/367 and VPA groups at 14 dpi. n = 3 mice per group. Scale bar: 50 μm.</p

MicroRNA-Mediated In Vitro and In Vivo Direct Conversion of Astrocytes to Neuroblasts

<div><p>Background</p><p>The conversion of astrocytes to neuroblasts holds great promise for treatment of neurodegenerative and traumatic brain diseases.</p><p>Methodology and Principal Findings</p><p>Here we have shown that adult human astrocytes could be reprogrammed to neuroblasts by miR-302/367, both in vivo and in vitro. However, the reprogramming of adult mouse astrocytes to neuroblasts required valproic acid (VPA), a histone deacetylase inhibitor. Following induction of astrocytes toward neurons the expression of pluripotency markers were not detected, which suggested direct cell conversion. We did not observed tumor formation during two months follow up.</p><p>Conclusions and Significance</p><p>These results show that neuroblasts can be generated directly from adult human and mouse astrocytes by miR-302/367-driven induction. This approach seems promising for converting glial scar cells into neuroblasts in a wide range of neurological diseases.</p></div

Administration of GFP and miR-302/367 expressing lentiviral particles into the striatum and the distribution of transduced cells.

<p>Transduction and continuous expression of vectors were confirmed by injecting a GFP expressing empty lentiviral vector and lentiviral particle that included the miR-302/367+GFP cluster. Scale bar: 100 μm.</p

Human astrocytes were transduced with the miR-302/367+GFP expressing vector in vitro and then transplanted into mice striata.

<p><b>(A)</b> Schematic diagram of the experiment. <b>(B)</b> Transfection efficiency. <b>(C)</b> A number of transplanted human astrocytes expressed neuroblasts and neuronal markers 9 days after transduction (7 days after transplantation). <b>(D)</b> Quantification of cells immunostained with anti-doublecortin (DCX), anti-TUJ1 and anti-NeuN antibodies. n = 3. Scale bar: 50 μm.</p

Induced neurons showed mature neuronal properties at six and ten weeks after transduction in vitro.

<p><b>(A)</b> The majority of neurons were positive for glutamate (Glu) as a marker for excitatory neurons at six weeks post-transduction. <b>(B)</b> Quantification of immunostaining data provided in A. <b>(C and D)</b> Whole cell patch clamp recording from induced neuron-like cells at six weeks post-transduction (n = 25) showed single action potential-like spikes. <b>(E)</b> Similar recording at ten weeks post-transduction (n = 10) showed repetitive spike firing. <b>(F)</b> Treatment with 1 μM TTX as sodium channel blocker, inhibited spike firing. **p< 0.01. Scale bar: 50 μm</p

miR-302/367 and valproic acid (VPA) converted the transducted cells into neuroblasts.

<p>Doublecortin (DCX⁺) cells were detected at 7 and 14 days post-injection (dpi) in animals pre-treated with VPA that afterwards received miR-302/367. n = 3 mice per group. Scale bar: 30 μm.</p

Determining of the fate of cells transduced with GFP expressing vector.

<p>(A) Immunofluorescence studies against astrocytes, oligodendrocytes, neurons and mature myelinating cell markers showed that GFAP⁺ astrocytes were the main population of transduced cells. (B) Quantification result of immunostaining against different markers<b>.</b> ***p<0.001, n = 3 mice per group. Scale bar: 50 μm.</p

Global, regional, and national burden of epilepsy, 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016

Global, regional, and national burden of epilepsy, 1990–2016: a systematic analysis for the Global Burden of Disease Study 201