Nuclear transcriptome profiling of induced pluripotent stem cells and embryonic stem cells identify non-coding loci resistant to reprogramming

<p>Identification of functionally relevant differences between induced pluripotent stem cells (iPSC) and reference embryonic stem cells (ESC) remains a central question for therapeutic applications. Differences in gene expression between iPSC and ESC have been examined by microarray and more recently with RNA-SEQ technologies. We here report an in depth analyses of nuclear and cytoplasmic transcriptomes, using the CAGE (cap analysis of gene expression) technology, for 5 iPSC clones derived from mouse lymphocytes B and 3 ESC lines. This approach reveals nuclear transcriptomes significantly more complex in ESC than in iPSC. Hundreds of yet not annotated putative non-coding RNAs and enhancer-associated transcripts specifically transcribed in ESC have been detected and supported with epigenetic and chromatin-chromatin interactions data. We identified super-enhancers transcriptionally active specifically in ESC and associated with genes implicated in the maintenance of pluripotency. Similarly, we detected non-coding transcripts of yet unknown function being regulated by ESC specific super-enhancers. Taken together, these results demonstrate that current protocols of iPSC reprogramming do not trigger activation of numerous <i>cis</i>-regulatory regions. It thus reinforces the need for already suggested deeper monitoring of the non-coding transcriptome when characterizing iPSC clones. Such differences in regulatory transcript expression may indeed impact their potential for clinical applications.</p

Number of patients with puboperineal muscle injury.

Number of patients with puboperineal muscle injury.</p

Identification of Licopyranocoumarin and Glycyrurol from Herbal Medicines as Neuroprotective Compounds for Parkinson's Disease

<div><p>In the course of screening for the anti-Parkinsonian drugs from a library of traditional herbal medicines, we found that the extracts of <i>choi-joki-to</i> and <i>daio-kanzo-to</i> protected cells from MPP⁺-induced cell death. Because <i>choi-joki-to</i> and <i>daio-kanzo-to</i> commonly contain the genus <i>Glycyrrhiza</i>, we isolated licopyranocoumarin (LPC) and glycyrurol (GCR) as potent neuroprotective principals from <i>Glycyrrhiza</i>. LPC and GCR markedly blocked MPP⁺-induced neuronal PC12D cell death and disappearance of mitochondrial membrane potential, which were mediated by JNK. LPC and GCR inhibited MPP⁺-induced JNK activation through the suppression of reactive oxygen species (ROS) generation, thereby inhibiting MPP⁺-induced neuronal PC12D cell death. These results indicated that LPC and GCR derived from <i>choi-joki-to</i> and <i>daio-kanzo-to</i> would be promising drug leads for PD treatment in the future.</p></div

Effects of NMB on the differentiation and survival of SH-SY5Y cells.

<p>(A) Effect of NMB on the differentiation of SH-SY5Y cells. SH-SY5Y cells were cultured in DMEM-F12 (1:1) containing 1% FBS in the presence of NMB (10⁻⁷ M) or NGF (25 ng/mL) for 3 days. SH-SY5Y cells cultured in DMEM-F12 (1:1) containing 1% FBS were used as negative controls (NC). Representative photographs of the neurites are shown. Scale bars are 50 μmeter. (B) Bar graphs comparing the length of neurites. One hundred of neurites were randomly selected in each group and the length of the neurites was measured. *: P<0.01 vs. NC. (C) Effect of NMB on the survival of SH-SY5Y cells. SH-SY5Y cells were cultured in serum-free DMEM-F12 (1:1) in the presence of NMB (10⁻⁸ M or 10⁻⁷ M) or NGF (25 ng/mL) for 2 days. SH-SY5Y cells cultured in serum-free DMEM-F12 (1:1) were used as negative controls (NC) and those cultured in DMEM-F12 (1:1) containing 5% FBS (5% FBS) were used as positive controls. The cell viability observed in SH-SY5Y cells cultured in DMEM-F12 (1:1) containing 5% FBS was calculated as 100%, and the relative viability is shown (n = 8 per group). * and **: P<0.05 and P<0.01, respectively vs. negative control.</p

Two herbal medicines, <i>daio-kanzo-to</i> and <i>choi-joki-to</i>, identified as neuroprotective agents in the course of screening.

<p>(<b>A</b>) NGF-differentiated PC12D cells were treated with 0.3 µM rotenone and herbal medicine extract for 48 h. Cell viability was evaluated by trypan blue dye exclusion assay. (<b>B</b>) NGF-differentiated PC12D cells were treated with various concentrations of <i>choi-joki-to</i> or <i>daio-kanzo-to</i> in the presence of 0.3 mM MPP⁺ for 48 h. Cell viability was evaluated by trypan blue dye exclusion assay. Values are the means of triplicate samples; bars, s.d. ^**<i>p</i><0.01 compared with MPP⁺ group cells.</p

Representative pictures of intact fascia, injured fascia, and injured muscle of the pubococcygeus and puboperineal muscles.

Yellow arrows indicate the area of injury.</p

Licopyranocoumarin and glycyrurol decreased MPP⁺-induced intracellular ROS generation.

<p>(<b>A</b>) NGF-differentiated PC12D cells were pre-incubated for 1 h with 3 µM licopyranocoumarin (LPC) or 3 µM glycyrurol (GCR), then treated with 0.3 mM MPP⁺ for 12 h. Then, the samples were loaded with 2.5 µM CM-H₂DCFDA and the fluorescence intensities were measured by flow cytometry. (<b>B</b>) The ratio of cells exhibiting ROS production was analyzed. Values are the means of four independent experiments; bars, s.d. ^##<i>p</i><0.01 compared with control cells. ^**<i>p</i><0.01 compared with MPP⁺ group cells.</p

Patient demographics.

Patient demographics.</p

Expression of NMB by AdNMB.

<p>(A) Expression of NMB protein by AdNMB. HEK293 cells plated on type I collagen-coated dishes, were infected with AdNMB. After 24 h, the medium was replaced with serum-free DMEM, and the medium was collected 2 hours later for ELISA (n = 6 per group). *: P<0.001 vs. AdGFP infection. (B) Expression of NMB mRNA in the penis after AdNMB injection. AdGFP and AdNMB were injected into the penis and total RNA was extracted 7 days after injection. Real time PCR analysis was performed in order to examine the expression of NMB mRNA in the penis (n = 5 per group). *: P<0.01 vs. AdGFP infection.</p

Fig 2 -

A) Boxplot of incontinence ratio 2 days after urethral catheter removal. X axis: magnitude of pubococcygeus muscle injury. B) Boxplot of incontinence ratio two days after urethral catheter removal. X axis: magnitude of puboperineal muscle injury.</p