Comparing The Effects of Small Molecules BIX-01294, Bay K8644, RG-108 and Valproic Acid, and Their Different Combinations on Induction of Pluripotency Marker-Genes by Oct4 in The Mouse Brain

Objective: Every cell type is characterized by a specific transcriptional profile together with a unique epigenetic landscape. Reprogramming factors such as Oct4, Klf4, Sox2 and c-Myc enable somatic cells to change their transcriptional profile and convert them to pluripotent cells. Small molecules such as BIX-01294, Bay K8644, RG-108 and valproic acid (VPA) are reported as effective molecules for enhancing induction of pluripotency in vitro, however, their effects during in vivo reprogramming are addressed in this experimental study. Materials and Methods: In this experimental study, Oct4 expressing lentiviral particles and small molecules BIX-01294, Bay K8644 and RG-108 were injected into the right ventricle of mice brain and VPA was systematically administered as oral gavages. Animals treated with different combinations of small molecules for 7 or 14 days in concomitant with Oct4 exogenous expression were compared for expression of pluripotency markers. Total RNA was isolated from the rims of the injected ventricle and quantitative polymerase chain reaction (PCR) was performed to evaluate the expression of endogenous Oct4, Nanog, c-Myc, klf4 and Sox2 as pluripotency markers, and Pax6 and Sox1 as neural stem cell (NSC) markers. Results: Results showed that Oct4 exogenous expression for 7 days induced pluripotency slightly as it was detected by significant enhancement in expression of Nanog (p<0.05). Combinatorial administration of Oct4 expressing vector and BIX-01294, Bay K8644 and RG-108 did not affect the expression of pluripotency and NSC markers, but VPA treatment along with Oct4 exogenous expression induced Nanog, Klf4 and c-Myc (p<0.001). VPA treatment before the induction of exogenous Oct4 was more effective and significantly increased the expression of endogenous Oct4, Nanog, Klf4, c-Myc (p<0.01), Pax6 and Sox1 (p<0.001). Conclusion: These results suggest VPA as the best enhancer of pluripotency among the chemicals tested, especially when applied prior to pluripotency induction by Oct4

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

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

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

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

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

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

Characterization of hiPSC-NPs in vitro.

<p>(A) Real-time PCR shows changes in the expression a set of neural progenitor (NP) gene markers. Data shows increased expression of <i>NESTIN</i>, <i>SOX1,</i> and <i>PAX6</i> relative to hiPSCs as averaged from three independent experiments. (B) The number of positive cells for NP markers was determined by flowcytometric analysis, which revealed a high percentage of NPs that expressed NESTIN, OTX 2, and SOX1. Sample FACS histograms are presented as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071855#pone.0071855.s001" target="_blank">Fig. S1</a>. (C) Immunofluorescence staining of hiPSC-NPs showed high expression levels of NESTIN, SOX1, PAX6, and OTX2. The blue stain represents nuclear counterstaining with DAPI. (D) Immunofluorescence staining following spontaneous differentiation of hiPSC-NPs. The representative micrographs of MAPII, TUJ1 as neural markers, and GFAP as a glial marker show a high percentage of neural differentiation. (E) Quantitative data for the percent of positive cells following spontaneous differentiation.</p

Engrafted Human Induced Pluripotent Stem Cell-Derived Anterior Specified Neural Progenitors Protect the Rat Crushed Optic Nerve

<div><p>Background</p><p>Degeneration of retinal ganglion cells (RGCs) is a common occurrence in several eye diseases. This study examined the functional improvement and protection of host RGCs in addition to the survival, integration and neuronal differentiation capabilities of anterior specified neural progenitors (NPs) following intravitreal transplantation.</p><p>Methodology/Principal Findings</p><p>NPs were produced under defined conditions from human induced pluripotent stem cells (hiPSCs) and transplanted into rats whose optic nerves have been crushed (ONC). hiPSCs were induced to differentiate into anterior specified NPs by the use of Noggin and retinoic acid. The hiPSC-NPs were labeled by green fluorescent protein or a fluorescent tracer 1,1′ -dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) and injected two days after induction of ONC in hooded rats. Functional analysis according to visual evoked potential recordings showed significant amplitude recovery in animals transplanted with hiPSC-NPs. Retrograde labeling by an intra-collicular DiI injection showed significantly higher numbers of RGCs and spared axons in ONC rats treated with hiPSC-NPs or their conditioned medium (CM). The analysis of CM of hiPSC-NPs showed the secretion of ciliary neurotrophic factor, basic fibroblast growth factor, and insulin-like growth factor. Optic nerve of cell transplanted groups also had increased GAP43 immunoreactivity and myelin staining by FluoroMyelin™ which imply for protection of axons and myelin. At 60 days post-transplantation hiPSC-NPs were integrated into the ganglion cell layer of the retina and expressed neuronal markers.</p><p>Conclusions/Significance</p><p>The transplantation of anterior specified NPs may improve optic nerve injury through neuroprotection and differentiation into neuronal lineages. These NPs possibly provide a promising new therapeutic approach for traumatic optic nerve injuries and loss of RGCs caused by other diseases.</p></div