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

1. HNSC differentiation on Three Dimensional (3D) Culture

NOTE: Isolate and derive hNSCs,from an ethical approved tissue, described in a previous publication². Please follow the ethical and institutional guidelines while following this procedure.

1.1) 3D Culture Using 12-well Plate

1. Use aseptic technique throughout the procedure. In a biological safety cabinet, re-hydrate scaffolds by adding 2 ml/well of 70% ethanol prepared using water for irrigation (WFIr). Avoid touching the scaffolds by dispensing the 70% ethanol down the wall of each well.
2. Carefully aspirate the 70% ethanol and immediately wash the scaffolds with 2 ml/well DMEM: F12 for 1 min. Repeat the wash procedure twice. Carefully aspirate the DMEM: F12 after the final wash.
3. Coat scaffolds by adding 2 ml/well of laminin (10 µg/ml) in DMEM: F12. Incubate plate at 37 °C in a 5% CO₂ humidified incubator for a minimum of 2 hr. Wash plate with warm DMEM: F12.
4. Seed each scaffold with approximately 500,000 hNSCs in a volume of 150 µl of RMM+GFs.
5. Incubate the plate for 3 hr at 37 °C in a 5% CO₂ humidified incubator to allow cells to settle into the scaffolds.
6. Add 3.5 ml of RMM to each well taking care not to dislodge cells from scaffolds.
7. Incubate the plate for 7 days; replace RMM medium (3.5 ml/well) after 1 day (first feeding) and again after 2-3 days from the first feeding.
8. After 7 days, remove medium and fix cells with 4% paraformaldehyde (PFA) for immunocytochemistry (ICC) or add 500 µl/well of lysis reagent for total RNA extraction (store plate at ≤ -80 °C or proceed to total RNA extraction).

1.2) Measurement of Axon Process Outgrowth

1. Stain 4% PFA fixed differentiated hNSCs according to a standard ICC protocol²⁰ using β3-tubulin (TUBB3) primary antibody detected with a fluorochrome conjugated secondary antibody. Counterstain cell nuclei with Hoechst (1 µM).
2. Capture representative images of β3-tubulin stained cells using a fluorescent microscope; save images as tiff or jpeg. Measure of axon process outgrowth using a software measurement tool (e.g. Image-Pro Plus).
3. Open the image, select measurements option in the tool bar, and select trace feature.
4. 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. Repeat to measure all axon outgrowthswith in the field of view.
5. Express measurements as pixels or µm (pending calibration set up). Export length measurements to a spreadsheet program for sample comparative and statistical analysis.

2. MiRNA Expression Using a Stem Cells and Developmental Pathways Focused miRNA PCR Array

2.1) miRNA total RNA extraction

1. Perform miRNA extraction on scaffolds and 2D grown samples (differentiated and undifferentiated, hNSC control).
2. Thaw scaffolds, inserted in a 12 well plate, if required. Combine lysis reagent contents (500 µl) of 2 wells in the same 1.5 ml Eppendorf tube, final volume 1 ml of Lysis reagent. Take care to leave the scaffold in the well.
3. For previously collected as dry cell pelleted 2D grown samples (differentiated hNSC 2D cultures and undifferentiated control) add 1 ml of lysis reagent and transfer contents into 1.5 ml microcentrifuge tube.
4. Homogenize each test sample using a 1 ml syringe and a short 20 to 25 gauge needle. Pass the lysate through the needle using a 1 ml syringe up to 5 times until a homogeneous lysate is achieved.
5. Add 200 µl of chloroform to each Eppendorf tube and cap securely. Invert and vortex tubes to mix contents.
6. Centrifuge each 1.5 ml microcentrifuge tube containing homogenate for 15 min at ≥12,000 x g.
7. Transfer the upper aqueous phase of each sample (avoid transferring any interphase, pink coloured liquid) to a fresh 1.5 ml microcentrifuge tube containing 750 µl of 100% ethanol. Mix thoroughly by inversion.
8. Pipette up to 700 μl of each test sample, including any precipitate, into a mini column in a 2 ml collection tube. Close the lid and centrifuge at ≥8,000 x g for approximately 30 sec at room temperature. Discard the flow-through. Repeat this step using the remainder of the sample.
9. Perform a DNA digest for 15 min.
10. Add 700 μl buffer RWT to the mini column and centrifuge for approximately 30 sec at ≥8,000 x g. Discard the flow-through.
11. Wash mini column by adding 500 μl buffer RPE and centrifuging for approximately 30 sec at ≥8,000 x g. Discard the flow-through. Repeat wash.Centrifuge at ≥12,000 x g for 1 min to dry the membrane further.
12. Elute total RNA in a 1.5 ml Eppendorf tube by adding 40 μl of RNase-free water to the mini column and centrifuging for 1 min at ≥8,000 x g. Measure total RNA concentration with the aid of an appropriate spectrophotometer.

2.2) miRNA cDNA Preparation

1. Prepare the reverse-transcription master mix. Each sample requires 4 µl of 5x miScript HiSpec buffer, 2 µl of 10x nucleics mix, 2 µl miScript reverse transcriptase mix.
2. Aliquot 8 µl of master mix in a PCR tube, add required volume of total RNA (250 ng), and add RNase-free water to a final volume of 20 µl.
3. Incubate for 60 min at 37 °C.Incubate for 5 min at 95 °C to inactivate miScript Reverse Transcriptase Mix.
4. Add 200 μl RNase-free water to each 20 μl reverse-transcription reaction, store at –20 °C, or proceed with real-time PCR immediately.

2.3) Stem Cells and Developmental Pathways Focused miRNA PCR Array

1. Prepare the PCR components mix in a 15 ml tube by adding 1375 µl of 2x SYBR green master mix, 100 µl miRNA cDNA preparation, 275 µl of 10x miScript Universal Primer, and 1000 µl of RNase-free water (total volume 2,750 µl).
2. Transfer PCR components mix on a PCR loading reservoir. Carefully remove the 96-well plate PCR array from its sealed bag.
3. Add 25 μl PCR components mix to each well of the PCR Array using an 8-channel pipettor, or a 12-channel pipettor using only 8 tips. Change pipet tips following each pipetting step to avoid cross-contamination between the wells.
4. Seal plate with optical adhesive film, and centrifuge for 1 min at 1,000 x g at room temperature (15–25 °C) to remove bubbles. Visually inspect the plate from underneath to ensure no bubbles are present in the wells.
5. Place the 96 well plate PCR array in the real-time cycler.
6. Program the real-time cycler accordingly: 1 cycle at 95 °C for 10 min, and 45 cycles for 15 sec at 95 °C, and 1 min at 60 °C set a single (fluorescence data collection). 
7. Export the Ct values for all wells to a blank Excel spreadsheet for use with PCR Array Data Analysis Template Excel and web-based software.
   NOTE: Software is available at www.SABiosciences.com/pcrarraydataanalysis.php.

3. Computational Analysis for miRNA Target Prediction and Validation of Target mRNAs by Reporter Plasmid Transfection and Ddual Luciferase Assay.

3.1) Computational Analysis for miRNA Target Prediction

1. Browse PicTar (http://pictar.mdc-berlin.de/)²¹, online available algorithm for the identification of miRNA target prediction mRNAs.
2. Click on "click here for PicTar prediction on vertebrates".  A new page will open.
3. In the new page, PicTar web interface, choose species in the drop down menu and insert miRNA of interest in the miRNA ID box. Click search for targets of a miRNA.
4. Observe a list of target mRNAs ranked by aPicTar score.
   NOTE: PicTar computes a score reflecting the likelihood that a given UTR will be targeted by the selected miRNA, and based on seed complementarity, thermodynamics and a combinatorial prediction for common targets in sets of co-expressed miRNAs²².
5. Similarly retrieve list of target mRNAs using DIANA-LAB (http://diana.cslab.ece.ntua.gr/microT/)²³, and TargetScan (http://www.targetscan.org/)²⁴, alternative on line available algorithms.
6. Compile a comparative table of miRNAs based on ranking obtained using different software tools. PicTar ranks by score, DianaLab by precision (based on signal to noise ratio and a precision score for the evaluation of the significance of the predicted results), and TargetScan by aggregate PCT (based on seed complementarity, thermodynamic free energy of binding, conservation over different species), see Table 1.

3.2) Validation of Target mRNAs by Reporter Plasmid Transfection and Dual Luciferase Assay.

NOTE: 3’ UTR luciferase reporters for the validation of target mRNA has been previously described²⁵.

1. Transfection
   1. Seed HeLa cells at a density of 10⁵ cells/well in 24-well plate.
   2. After 24 hr, co-transfect HeLa cells with 1.5 µl of transfection reagent and either 100 ng of NR4A3 3’ UTRor SOX5 3’ UTR plasmid, as well as 20 nM miRNA mimics (hsa-miR-96-5p for SOX5 3’ UTR, and hsa-miR-7-5p, and hsa-miR-17-5p for NR4A3 3’ UTR respectively) per well.
   3. Co-transfect control wells with NR4A3 3’ UTRor SOX5 3’ UTRplasmid and fluorochrome labelled negative control siRNAs. The transfection of control wells with fluorochrome labelled negative control siRNAs allows evaluation of transfection efficiency and accounts for mimic miRNA 3’UTR bind specificity.
2. Measurement of Dual Luciferase Activities
   1. Measure Firefly and Renilla luciferase activities 24 hr post transfection. Remove cell growth medium.
   2. Prepare working solution I by adding 50 µl of substrate I into 10 ml of Solution I; mix thoroughly. Add 300 µl of working solution I into each 24 well.
   3. Transfer the contents of each 24 well into 3 wells of a 96 white plate, 100 µl each. Wait 10 min and measure Firefly luciferase in luminometer set for luminescence acquisition.
   4. Prepare working solution II by adding 50 µl of substrate II into 10 ml of solution II; mix thoroughly. Add 100 µl of working solution I into each 96 well already containing 100 µl of working solution I.
   5. Wait 10 min and measure Renilla luciferase in luminometer set for luminescence acquisition. Calculate the ratio of luminescence from the Firefly luciferase to the Renilla luciferase.