Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

The overall goal of the following experiment is to assess differentiation micro RNA, profiling and target Mr.NA validation of a human neural stem cell line. This is accomplished by first culturing human neural stem cells on a three dimensional culture system to induce cell differentiation. The second step is to quantify differentiation by measuring axon process outgrowth.

Next, changes in micro RNA expression are evaluated by micro R-N-A-P-C-R array. Computational analysis of selected differentially expressed microRNAs is then performed to identify a set of putative target mRNAs. Ultimately, the target mRNAs are validated by reporter plasmid, transfection, and a dual luciferase assay.

So the method to be presented here can help answer key issues in the neural stem cell field, such as the characterization of expressed microRNAs, and they target messenger RNAs in human neural stem cells when they're undergoing differentiation. Though this method can provide insight into neural stem cell biology. It can also be applied to other stem cell systems such as mesenchymal stem cells, induced pluripotent stem cells, or other systems that require induction of differentiation and molecular characterization.

Visual demonstration of this method breaks down the critical steps in the differentiation of a human neuros stem cell line and investigation of microRNA profiling and the validation of target mRNA demonstrating part of the procedure will be lavania, a scientist from my laboratory. This entire procedure should be performed in a biological safety cabinet using aseptic techniques. Rehydrate the scaffolds by adding to each well of the AL VX 12.

Well plate two milliliters of 70%ethanol prepared using water for irrigation. Avoid touching the scaffolds by dispensing the 70%ethanol down the wall of each. Well carefully aspirate the 70%ethanol and immediately wash the scaffolds with two milliliters per well of D-M-E-M-F 12 for one minute.

Repeat the wash procedure twice carefully aspirate the D-M-E-M-F 12 after the final wash. Coat the scaffolds by adding two milliliters of laminin in D-M-E-M-F 12 to each. Well incubate the plate at 37 degrees Celsius in a 5%carbon dioxide humidified incubator for a minimum of two hours.

Next, wash the plate with warm D-M-E-M-F 12 seed each scaffold with approximately 500, 000 human neural stem cells or H nscs in a volume of 150 microliters of RMM medium with growth factors. Incubate the plate for three hours to allow the cells to settle into the scaffolds after three hours. Add 3.5 milliliters of RMM to each well Taking care not to dislodge cells from the scaffolds.

Incubate the plate for seven or 21 days. Replace the RMM medium after one day, which is the first feeding and replace the medium again two to three days after the first feeding. Seven and 21 days after the 3D culture of H nscs.

Remove the RMM medium and fix the differentiated cells with 4%paraform aldehyde. The fixed cells are then stained using a beta three tubulin primary antibody detected with a fluorochrome conjugated. Secondary antibody cell nuclei are counterstain with hooks.

Capture representative images of the beta three tubulin stained cells. Using a fluorescent microscope, save the images in TIFF or JPEG format. The measurement of axon process outgrowth is accomplished using the image PRO plus seven software measurement tool.

To begin open the image, select the measurement option in the toolbar and select trace feature. To start the trace, select the start point on the axon outgrowth by clicking the mouse button trace along the entire length of the axon outgrowth right click to finish each trace. In this manner, measure all axon outgrowths within the field of view express measurements as pixels or micrometers.

Export length measurements to a spreadsheet program for sample comparative and statistical analysis to evaluate changes in micro RNA expression of the three dimensionally cultured HSCs. First perform micro RNA extraction and micro RNA CD NA preparation as described in the accompanying protocol. Text immediately after the micro RNA CD NA is obtained.

Prepare the PCR components mixture for the real-time PCR in a 15 milliliter tube. Add 1375 microliters of a two X-S-Y-B-R green master mix. 100 microliters of micro RNA CD NA 275 microliters of the universal primer and 1000 microliters of RNAs free water transfer.

The PCR components mix to a PCR loading reservoir. Carefully remove the 96 well plate PCR array from its sealed bag. Using a multi-channel pipetter, add 25 microliters of the PCR components.

Mix to each well of the PCR array. Change the pipette tips following each pipetting step to avoid cross contamination between the wells seal. The 96 well plate with optical adhesive film and centrifuge for one minute at 1000 GS at room temperature to remove bubbles visually inspect the plate to ensure no bubbles are present in the wells place.

The 96 well plate PCR array in the realtime cycler program. The realtime cycler accordingly. Subsequently, export the CT values for all wells to a blank Excel spreadsheet for PCR array Data analysis, an algorithm that is available online PIC ktar is used for the identification of predicted micro RNA targets.

Click on, click here for pic ktar prediction on vertebrates to open a new page in the PIC KTAR web interface. Choose species in the dropdown menu and insert the micro RNA of interest in the micro RNA ID box. Click search for targets of a micro RNA.

A list of target mRNAs ranked by a PIC KTAR score will be presented. The rankings obtained are then used to compile a comparative table of microRNAs. A dual luciferase reporter plasmid is used as a tool to validate the target mRNAs firefly and renal luciferase activities are measured 24 hours after the transfection of hela cells.

Prepare working solution one by adding 50 microliters of substrate, one into 10 milliliters of solution one and mixing thoroughly remove cell growth medium from each well of the 24 well plate and add 300 microliters of working solution one to each. Well transfer 100 microliters of the contents of each well. Of the 24 well plate to three wells of a 96 well plate wait for 10 minutes and then measure firefly luciferase activity in a luminometer set for luminescence acquisition.

Prepare working solution two by adding 50 microliters of substrate, two to 10 milliliters of solution two and mixing thoroughly. Add 100 microliters of working solution two into each well of the 96 well plate already containing 100 microliters of working solution. One wait for 10 minutes and then measure renal el luciferase activity.

Subsequently, the ratio of luminescence from the firefly luciferase to the renal luciferase is calculated. Representative micro photographs of H NSCS on 3D scaffolds were acquired following immunochemistry staining for beta three tubulin shown in green and nuclei counter stain with hooks shown in blue measurement of axon processes was performed with the aid of image analysis software. A comparison of axon process outgrowth between H nscs grown with 3D scaffolds and with traditional flat surface or 2D cultures shows that at one week or three weeks the 3D differentiated HSCs exhibited greater axon process outgrowth.

Computational analysis for target prediction resulted in the information presented here a list of microRNAs predicted target genes and precision score and aggregate values. SOX five and NR four A three were identified as putative target genes. A dual luciferase reporter assay was used to validate the targets of the microRNAs as shown here.

Relative luciferase activity from human three prime UTR reporters of SOX five and NR four A three genes was significantly knocked down in the presence of the indicated microRNAs. After watching this video, you should have a good understanding of how to differentiate him in neural stem cells and investigate micro RNA expression and validation of target Messenger.RNA.