Implantation of neural stem cells embedded in hyaluronic acid and collagen composite conduit promotes regeneration in a rabbit facial nerve injury model

The implantation of neural stem cells (NSCs) in artificial scaffolds for peripheral nerve injuries draws much attention. NSCs were ex-vivo expanded in hyaluronic acid (HA)-collagen composite with neurotrophin-3, and BrdU-labeled NSCs conduit was implanted onto the ends of the transected facial nerve of rabbits. Electromyography demonstrated a progressive decrease of current threshold and increase of voltage amplitude in de-innervated rabbits after implantation for one, four, eight and 12 weeks compared to readouts derived from animals prior to nerve transection. The most remarkable improvement, observed using Electrophysiology, was of de-innervated rabbits implanted with NSCs conduit as opposed to de-innervated counterparts with and without the implantation of HA-collagen, NSCs and HA-collagen, and HA-collagen and neurotrophin-3. Histological examination displayed no nerve fiber in tissue sections of de-innervated rabbits. The arrangement and S-100 immunoreactivity of nerve fibers in the tissue sections of normal rabbits and injured rabbits after implantation of NSCs scaffold for 12 weeks were similar, whereas disorderly arranged minifascicles of various sizes were noted in the other three arms. BrdU+ cells were detected at 12 weeks post-implantation. Data suggested that NSCs embedded in HA-collagen biomaterial could facilitate re-innervations of damaged facial nerve and the artificial conduit of NSCs might offer a potential treatment modality to peripheral nerve injuries

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Infrared spectroscopic identification of patients with Beta-Thalassemia

Background: The aim of this study was to investigate the potential of infrared (IR) spectroscopy as a fast and reagent-free adjunct tool in the diagnosis and screening of \u3b2-thalassemia. Methods: Blood was obtained from 56 patients with \u3b2-thalassemia major, 1 patient with hemoglobin H disease, and 35 age-matched controls. Hemolysates of blood samples were centrifuged to remove stroma. IR absorption spectra were recorded for duplicate films dried from 5 \u3bcL of hemolysate. Differentiation between the two groups of hemoglobin spectra was by two statistical methods: an unsupervised cluster analysis and a supervised linear discriminant analysis (LDA). Results: The IR spectra revealed changes in the secondary structure of hemoglobin from \u3b2-thalassemia patients compared with that from controls, in particular, a decreased \u3b1-helix content, an increased content of parallel and antiparallel \u3b2-sheets, and changes in the tyrosine ring absorption band. The hemoglobin from \u3b2-thalassemia patients also showed an increase in the intensity of the IR bands from the cysteine 12SH groups. The unsupervised cluster analysis, statistically separating spectra into different groups according to subtle IR spectral differences, allowed separation of control hemoglobin from \u3b2-thalassemia hemoglobin spectra, based mainly on differences in protein secondary structure. The supervised LDA method provided 100% classification accuracy for the training set and 98% accuracy for the validation set in partitioning control and \u3b2-thalassemia samples. Conclusion: IR spectroscopy holds promise in the clinical diagnosis and screening of \u3b2-thalassemia.Peer reviewed: YesNRC publication: Ye

Induction and isolation of hNCSCs from hESCs.

<p><b>(A):</b> A colony of H9 hESCs double stained for p75 (green) and HNK-1 (red) after one week of culture on PA6. Note that the expression of p75 and HNK-1 is outside or at the edge of the hESC colony, scale bar = 200μm; <b>(B):</b> Reverse transcription-polymerase chain reaction analysis (RT-PCR) of <i>p75</i> expression, note that the expression of <i>p75</i> is gradually increased up to day 10; <b>(C):</b> Analysis of H9-derived p75⁺ and HNK1⁺ cells by FACS analysis. The dot plots are representative of three independent experiments; <b>(D):</b> Different gene expression profiles are displayed in p75⁺ and p75^- populations as assessed by RT-PCR. Note that p75⁺ cells express markers of NCSC and sympathetic neurons. The image is the representative of three independent experiments.</p

<i>MycN</i> Is Critical for the Maintenance of Human Embryonic Stem Cell-Derived Neural Crest Stem Cells

<div><p>The biologic studies of human neural crest stem cells (hNCSCs) are extremely challenging due to the limited source of hNCSCs as well as ethical and technical issues surrounding isolation of early human embryonic tissues. On the other hand, vast majority of studies on <i>MycN</i> have been conducted in human tumor cells, thus, the role of <i>MycN</i> in normal human neural crest development is completely unknown. In the present study, we determined the role of <i>MycN</i> in hNCSCs isolated from <i>in vitro</i>-differentiating human embryonic stem cells (hESCs). For the first time, we show that suppression of <i>MycN</i> in hNCSCs inhibits cell growth and cell cycle progression. Knockdown of <i>MycN</i> in hNCSCs increases the expression of <i>Cdkn1a</i>, <i>Cdkn2a</i> and <i>Cdkn2b</i>, which encodes the cyclin-dependent kinases p21^CIP1, p16 ^INK4a and p15^INK4b. In addition, <i>MycN</i> is involved in the regulation of human sympathetic neurogenesis, as knockdown of <i>MycN</i> enhances the expression of key transcription factors involved in sympathetic neuron differentiation, including <i>Phox2a</i>, <i>Phox2b</i>, <i>Mash1</i>, <i>Hand2</i> and <i>Gata3</i>. We propose that unlimited source of hNCSCs provides an invaluable platform for the studies of human neural crest development and diseases.</p></div

<i>MycN</i> knockdown affects the expression of cell cycleand autonomic neuron development-related genes.

<p>FACS-sorted p75⁺ hNCSCs were transduced with concentrated pGLVH1/GFP-MYCN shRNA virus and selected in puromycin for two weeks.<b>(A):</b> Real-time PCR analysis showing the expression of cell cycle-related genes was altered by <i>MycN</i> suppression, *p<0.05, ***p<0.001; ****p<0.0001; quantitative data was acquired from three independent experiments;<b>(B):</b> Western blot shows knockdown of <i>MycN</i> increases the expression of p15, whereas decreases the expression of Cyclin D1; <b>(C)</b> Real-time PCR analysis showing the expression of polycomb genes and autonomic neuronal markers was altered by <i>MycN</i> suppression, *p<0.05, **p<0.01;***p<0.001; ****p<0.0001, quantitative data were acquired from three independent experiments.</p

Induction of neural crest cells from hESCs.

<p>For neural crest induction, H9 cells were plated at 5-10x10²/cm² on a confluent layer of PA6 cells in 6 well plates or chamber slides in induction medium as described in <i>Materials and Methods</i>. <b>(A):</b>Phase contrast photographs of neural crest differentiation of H9 induced by PA6, scale bar = 200μm;<b>(B):</b>Dynamic expression of sympathetic neuronal markers, <i>c-Myc</i> and <i>MycN</i> in NC differentiating cells from hESCs as determined by PCR. The image is the representative of three independent experiments.</p

<i>MycN</i> knockdown inhibits cell growth in hNCSCs.

<p>FACS-sorted p75⁺ hNCSCs were plated at a cell density of 4×10³ cells/cm² in self-renewal media on 6-well plates that were pre-coated with 15 μg/ml polyornithine, 1 μg/ml laminin and 10 μg/ml fibronectin for 24 hours. Freshly isolated hNCSCs were transduced with concentrated pGLVH1/GFP-<i>MycN</i>shRNA virus and selected in puromycin (2 μg/ml) for two weeks, as described in Materials and Methods.<b>(A):</b> The GFP expression in control and <i>MycN</i>-transduced hNCSCs; <b>(B):</b> FACS analysis shows the similar transduction efficiency in control and <i>MycN</i>-transduced hNCSCs; <b>(C):</b> RT-PCR analysis showed the expression of <i>MycN</i> in control and <i>MycN</i>kd hNCSCs after 2 weeks of antibiotics selection. Image shown is the representative of three independent experiments; <b>(D):</b> Freshly sorted cells were plated onto 15 μg/ml polyornithine, 1 μg/ml laminin and 10 μg/ml fibronectin coated plates at a concentration of 10x10³ cells/well and grown in NC media. Within two days to adherent plate, <i>MycN</i> knocked down cells initiated a change from small, NCSC-like to a larger shape.</p