We acknowledge Julie Heward for helping in the preparation of hNSCs, and Lavaniya Thanabalasundaram for her technical support with HeLa culturing and transfection.

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&#160;12-well Plate
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Coat scaffolds by adding 2 ml/well of laminin (10 &#181;g/ml) in DMEM: F12. Incubate plate at 37 &#176;C in a 5% CO2 humidified incubator for a minimum of 2 hr. Wash plate with warm DMEM: F12.</li>
	<li>Seed each scaffold with approximately 500,000 hNSCs in a volume of 150 &#181;l of RMM+GFs.</li>
	<li>Incubate the plate for 3 hr at 37 &#176;C in a 5% CO2 humidified incubator to allow cells to settle into the scaffolds.</li>
	<li>Add 3.5 ml of RMM to each well taking care not to dislodge cells from scaffolds.</li>
	<li>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.</li>
	<li>After 7 days, remove medium and fix cells with 4% paraformaldehyde (PFA) for immunocytochemistry (ICC) or add 500 &#181;l/well of lysis reagent for total RNA extraction (store plate at &#8804; -80 &#176;C or proceed to total RNA extraction).</li>
</ol>
1.2) Measurement of Axon Process Outgrowth
<ol>
	<li>Stain 4% PFA fixed differentiated hNSCs according to a standard ICC protocol20 using &#946;3-tubulin (TUBB3) primary antibody detected with a fluorochrome conjugated secondary antibody. Counterstain cell nuclei with Hoechst (1 &#181;M).</li>
	<li>Capture representative images of &#946;3-tubulin stained cells using a fluorescent microscope; save images as tiff or jpeg. Measure of axon process outgrowth using a&#160;software&#160;measurement tool (e.g. Image-Pro Plus).</li>
	<li>Open the image, select measurements option in the tool bar, and select trace feature.</li>
	<li>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.</li>
	<li>Express measurements as pixels or &#181;m (pending calibration set up). Export length measurements to a spreadsheet program for sample comparative and statistical analysis.</li>
</ol>
2. MiRNA Expression Using a Stem Cells and Developmental Pathways Focused miRNA PCR Array
2.1) miRNA total RNA extraction
<ol>
	<li>Perform miRNA extraction on scaffolds and 2D grown samples (differentiated and undifferentiated, hNSC control).</li>
	<li>Thaw scaffolds, inserted in a 12 well plate, if required. Combine lysis reagent contents (500 &#181;l) 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Add 200 &#181;l of chloroform to each Eppendorf tube and cap securely. Invert and vortex tubes to mix contents.</li>
	<li>Centrifuge each 1.5 ml microcentrifuge tube containing homogenate for 15 min at &#8805;12,000 x g.</li>
	<li>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 &#181;l of 100% ethanol. Mix thoroughly by inversion.</li>
	<li>Pipette up to 700 &#956;l of each test sample, including any precipitate, into a mini column in a 2 ml collection tube. Close the lid and centrifuge at &#8805;8,000 x g for approximately 30 sec at room temperature. Discard the flow-through. Repeat this step using the remainder of the sample.</li>
	<li>Perform a DNA digest for 15 min.</li>
	<li>Add 700 &#956;l buffer RWT to the mini column and centrifuge for approximately 30 sec at &#8805;8,000 x g. Discard the flow-through.</li>
	<li>Wash mini column by adding 500 &#956;l buffer RPE and centrifuging for approximately 30 sec at &#8805;8,000 x g. Discard the flow-through. Repeat wash.Centrifuge at &#8805;12,000 x g for 1 min to dry the membrane further.</li>
	<li>Elute total RNA in a 1.5 ml Eppendorf tube by adding 40 &#956;l of RNase-free water to the mini column and centrifuging for 1 min at &#8805;8,000 x g. Measure total RNA concentration with the aid of an appropriate spectrophotometer.</li>
</ol>
2.2) miRNA cDNA Preparation
<ol>
	<li>Prepare the reverse-transcription master mix. Each sample requires 4 &#181;l of 5x miScript HiSpec buffer, 2 &#181;l of 10x nucleics mix, 2 &#181;l miScript reverse transcriptase mix.</li>
	<li>Aliquot 8 &#181;l 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 &#181;l.</li>
	<li>Incubate for 60 min at 37 &#176;C.Incubate for 5 min at 95 &#176;C to inactivate miScript Reverse Transcriptase Mix.</li>
	<li>Add 200 &#956;l RNase-free water to each 20 &#956;l reverse-transcription reaction, store at &#8211;20 &#176;C, or proceed with real-time PCR immediately.</li>
</ol>
2.3) Stem Cells and Developmental Pathways Focused miRNA PCR Array
<ol>
	<li>Prepare the PCR components mix in a 15 ml tube by adding 1375 &#181;l of 2x SYBR green master mix, 100 &#181;l miRNA cDNA preparation, 275 &#181;l of 10x miScript Universal Primer, and 1000 &#181;l of RNase-free water (total volume 2,750 &#181;l).</li>
	<li>Transfer PCR components mix on a PCR loading reservoir. Carefully remove the 96-well plate PCR array from its sealed bag.</li>
	<li>Add 25 &#956;l 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.</li>
	<li>Seal plate with optical adhesive film, and centrifuge for 1 min at 1,000 x g at room temperature (15&#8211;25 &#176;C) to remove bubbles. Visually inspect the plate from underneath to ensure no bubbles are present in the wells.</li>
	<li>Place the 96 well plate PCR array in the real-time cycler.</li>
	<li>Program the real-time cycler accordingly: 1 cycle at 95 &#176;C for 10 min, and 45 cycles for 15 sec at 95 &#176;C, and 1 min at 60 &#176;C set a single (fluorescence data collection).&#160;</li>
	<li>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 <a href="http://www.SABiosciences.com/pcrarraydataanalysis.php">www.SABiosciences.com/pcrarraydataanalysis.php</a>.</li>
</ol>
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
<ol>
	<li>Browse PicTar (<a href="http://pictar.mdc-berlin.de/">http://pictar.mdc-berlin.de/</a>)21, online available algorithm for the identification of miRNA target prediction mRNAs.</li>
	<li>Click on &#34;click here for PicTar prediction on vertebrates&#34;.&#160; A&#160;new page will open.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Similarly retrieve list of target mRNAs using DIANA-LAB (<a href="http://diana.cslab.ece.ntua.gr/microT/">http://diana.cslab.ece.ntua.gr/microT/</a>)23, and TargetScan (<a href="http://www.targetscan.org/">http://www.targetscan.org/</a>)24, alternative on line available algorithms.</li>
	<li>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.</li>
</ol>
3.2) Validation of Target mRNAs by Reporter Plasmid Transfection and Dual Luciferase Assay.
NOTE: 3&#8217; UTR luciferase reporters for the validation of target mRNA has been previously described25.
<ol>
	<li>Transfection
	<ol>
		<li>Seed HeLa cells at a density of 105 cells/well in 24-well plate.</li>
		<li>After 24 hr, co-transfect HeLa cells with 1.5 &#181;l of transfection reagent and either 100 ng of NR4A3 3&#8217; UTRor SOX5 3&#8217; UTR plasmid, as well as 20 nM miRNA mimics (hsa-miR-96-5p for SOX5 3&#8217; UTR, and hsa-miR-7-5p, and hsa-miR-17-5p for NR4A3 3&#8217; UTR respectively) per well.</li>
		<li>Co-transfect control wells with NR4A3 3&#8217; UTRor SOX5 3&#8217; 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&#8217;UTR bind specificity.</li>
	</ol>
	</li>
	<li>Measurement of Dual Luciferase Activities
	<ol>
		<li>Measure Firefly and Renilla luciferase activities 24 hr post transfection. Remove cell growth medium.</li>
		<li>Prepare working solution I by adding 50 &#181;l of substrate I into 10 ml of Solution I; mix thoroughly. Add 300 &#181;l of working solution I into each 24 well.</li>
		<li>Transfer the contents of each 24 well into 3 wells of a 96 white plate, 100 &#181;l each. Wait 10 min and measure Firefly luciferase in luminometer set for luminescence acquisition.</li>
		<li>Prepare working solution II by adding 50 &#181;l of substrate II into 10 ml of solution II; mix thoroughly. Add 100 &#181;l of working solution I into each 96 well already containing 100 &#181;l of working solution I.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

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All authors are employees, stock and/or stock option holders in ReNeuron Ltd or its parent company.&#160;This study was supported by ReNeuron (RENE.L).&#160;

The development of new in vitro assays to assess and improve the differentiation of stem cells has always presented a challenge for the investigation of their functionalities and therapeutic capabilities. Long term and stable attachment of hNSCs, required for the development of a robust 3D in vitro differentiation assay, was addressed by testing several 3D substrates with different characteristics in term of materials and structures. As part of the assay development we also assessed optimal cell seeding concentration and identified the requirement for the scaffolds to be laminin coated. Although our hNSCs can spontaneously differentiate upon 4-OHT and growth factor removal, the implementation of a 3D culture system showed a significant improvement in their differentiation capability as measured by axon process outgrowth when compared to standard 2D differentiated hNSCs.
To investigate the effect of 3D induced differentiation on hNSC miRNA expression we used a PCR array. Compared with standard chip microarray PCR array offers the advantages of a direct quantification and validation of miRNA of interest. Although the PCR array is limited in term of total number of miRNA per array, for example in here less than 96, it can be tailored to specifically include miRNAs of interest, in our case previously reported in stem cell development. The expression of pathway focused miRNAs was profiled in 3D and 2D differentiated hNSCs compared with undifferentiated hNSCs. The most significantly down-regulated miRNAs were selected and analyzed to assess their putative target mRNAs with the intent of determining their functionality and biological interpretation using online available algorithms (DIANA Lab, PicTar, and TargetScans).
A single miRNA can recognize hundreds of targets and for this reason we validated miRNA interactions with 3&#8217; UTR mRNAsthat may be suitable candidates in axonal regulation. NR4A3, a target of the hsa-miR-7, and miR-17 family, is a member of the NR4A family nuclear receptors which, depending on their level of expression, are involved in the differentiation, survival, apoptosis and regulation of hippocampal axon guidance26. Similarly SOX5, a target of hsa-miR-96, is reported to control cell cycle progression in neural progenitors27axon length28, migration, post-migratory differentiation, and projections of neurons29. Both SOX5 and NR4A3 were identified as a direct target mRNA of hsa-miR-96, and hsa-miR-7 and 17 respectively by dual luciferase reporter assays. Dual luciferase (Firefly and Renilla) relies on two different reporter genes in the same plasmid and offers an improved system allowing normalization for effects caused by suboptimal transfection and cytotoxic effects compared with a single luciferase plasmid system. As part of our assay development we also optimized our transfection conditions in order to obtain high efficiency.
The protocolsdescribed herein may be exploited to further optimize and characterize in vitro hNSC differentiation which may beimplemented intransplantation protocols for pre-clinical studies and future clinical applications.

Stem cell research plays a central role in regenerative medicine, which also spans the disciplines of tissue engineering, developmental cell biology, cellular therapeutics, gene therapy, and biomaterials (scaffolds and matrices). Stem cells are important in both organ developmentand tissue repair; understanding how stem cells work is pivotal for developing new stem cell based treatments1. CTX0E03&#160;is a clonal, conditionally immortalized human neural stem cell (hNSC) line2&#160;isolated from first trimester human fetal cortex. CTX0E03 has defined quality characteristics that are essential for clinical cell banking under good manufacturing practice (GMP)3. HNSCs can spontaneously differentiate into neurons, glia, and oligodendrocytes and have been proven to ameliorate neurological deficits when transplanted in a rodent model of focal ischemia2,4,5. The CTX0E03 hNSCs are currently in clinical trials for stroke disability (NCT011511246 and NCT02117635, Clinicaltrials.gov).
MiRNAs have an average 22 nucleotides in length and are proven regulatory molecules7, they do not encode proteins, but rather bind 3 prime untranslated region (3&#8217;UTR) of mRNAs, regulating their stability and translation into proteins. MiRNAs are reported to participate in the regulation of a number of cellular processes, including development, proliferation, and differentiation8. Several miRNAs have been implicated in regulating the fate and specification of neural stem cells9.
In two dimensional (2D) standard culture environment the complexity of the in vivo environment is not matched, and consequently the induction and regulation of hNSC differentiation is not optimal. In vivo, cells are surrounded by a three dimensional (3D) microenvironment composed by other cells and extracellular ligands, including many types of collagens, laminin, and other matrix proteins (extracellular matrix, ECM). Previous studies have reported that the architectural topography of the materials mimicking ECMs and their geometry influence cell phenotype and fate10-15. A number of commercially available 3D scaffolds are available; we used a highly porous polystyrene scaffold engineered with a well-defined and uniform architecture into a 200 &#181;m thick membrane which provides a 3D space into which cells can invade and differentiate16-18.
Herein 2D and 3D differentiation assays are used to assess axonprocess outgrowth. Furthermore, we describe a method to profile the miRNA expression of a clinical grade hNSC line to further investigate miRNA effects on its differentiation. The methods in here illustrated are based on the ones originally described in19.

<table border="0" cellpadding="0" cellspacing="0" width="573">
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			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM:F12</td>
			<td>Invitrogen</td>
			<td>21331-020</td>
			<td>For RMM medium preparation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human Albumin Solution&#160;</td>
			<td>Baxter health care limited&#160;</td>
			<td>1501052</td>
			<td>For RMM medium used at a final concentration of&#160; 0.03%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transferrin, Human recombinant</td>
			<td>Sigma</td>
			<td>T8158</td>
			<td>For RMM medium used at a final concentration of&#160; 5 &#181;g/ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Putrescine DiHCl&#160;</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td>For RMM medium used at a final concentration of&#160; 16.2 &#181;g/ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin, Human recombinant&#160;&#160;</td>
			<td>Sigma</td>
			<td>I9278</td>
			<td>For RMM medium used at a final concentration of&#160; 5 &#181;g/ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Progesterone&#160;</td>
			<td>Sigma</td>
			<td>P6149</td>
			<td>For RMM medium used at a final concentration of&#160; 60 ng/ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine&#160;</td>
			<td>Invitrogen</td>
			<td>25030-24</td>
			<td>For RMM medium used at a final concentration of&#160; 2 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Selenite&#160;&#160;</td>
			<td>Sigma</td>
			<td>S9133</td>
			<td>For RMM medium used at a final concentration of 40 ng/ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">bFGF&#160;&#160;&#160;&#160;&#160;</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td>To be added to RMM medium, used at a final concentration of 10 ng/ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGF&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Peprotech</td>
			<td>AF-100-188</td>
			<td>To be added to RMM medium, used at a final concentration of 20 ng/ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-OHT</td>
			<td>Sigma</td>
			<td>H7904</td>
			<td>To be added to RMM medium, used at a final concentration of 100 nM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laminin</td>
			<td>AMS Biotech</td>
			<td>3400-010-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TrypZean/EDTA</td>
			<td>Lonza&#160;</td>
			<td>BE02-034E</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water for irrigation</td>
			<td>Baxter Healthcare</td>
			<td>UKF7114</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Defined Trypsin Inhibitor (DTI) (store -20&#176;C)</td>
			<td>Invitrogen</td>
			<td>R007-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alvetex 12 well plate&#160;</td>
			<td>Reinnervate Ltd</td>
			<td>AVP002</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol&#160;</td>
			<td>Merck&#160;</td>
			<td>1.00986.1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#946;III tubulin antibody</td>
			<td>Sigma</td>
			<td>T8660</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hoechst</td>
			<td>Sigma</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">miRNeasy mini kit</td>
			<td>Qiagen&#160;</td>
			<td>217004</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNase free DNase&#160;</td>
			<td>Qiagen&#160;</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chloroform</td>
			<td>Sigma</td>
			<td>C7559-5VL</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">miScript II RT Kit&#160;</td>
			<td>Qiagen&#160;</td>
			<td>218160</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SYBR green PCR kit</td>
			<td>Qiagen&#160;</td>
			<td>218073</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Development &#38; Differentiation miScript miRNA PCR Array</td>
			<td>Qiagen/ SABiosciences</td>
			<td>MIHS-103Z</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lightcycler 480 white 96 plate</td>
			<td>Roche</td>
			<td>&#160;4729692001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lightcycler 480&#160; sealing foil</td>
			<td>Roche</td>
			<td>&#160;4729757001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668-027</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vector NR4A3 miRNA 3&#39; UTR target clones&#160;</td>
			<td>GeneCopeia</td>
			<td>HmiT019538-MT0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vector SOX5 miRNA 3&#39; UTR target clones&#160;</td>
			<td>GeneCopeia</td>
			<td>HmiT017632-MT01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Allstars negative control siRNA AF 488 (Qiagen).</td>
			<td>Qiagen&#160;</td>
			<td>1027284</td>
			<td>MiRNA transfection control</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Luc-Pair miR Luciferase Assay</td>
			<td>GeneCopeia</td>
			<td>LPFR-M010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lightcycler 480&#160;</td>
			<td style="width:71px;">Roche</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">GloMax 96 microplate luminometer&#160;</td>
			<td style="width:71px;">Promega</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

differentiation of a human neural stem cell line on three dimensional cultures, analysis of microrna and putative target genes

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability (NCT01151124 and NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycERTAMconstruct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3&#8217;UTRs by dual reporter plasmid transfection and dual luciferase assay.

In Figure 1 is visualized the investigation and quantification of hNSC differentiation on 3D scaffolds compared with traditional flat surface cultures (2D) and expressedas axon process outgrowth measurements. In Figure 2 is representedthe workflow and the results for the miRNA quantification using stem cells and developmental pathways focused miRNA PCR array to investigate differential expression of miRNAs in 2D and 3D differentiated hNSCs compared with undifferentiated control. Following miRNA comparative analysis between differentiated and undifferentiated hNSCs, and computational analysis for target prediction, SOX5 and NR4A3 (NOR1) were identified as putative target mRNAs. To study the direct interaction between the miRNAs and their putative site on the 3&#8217;UTR mRNA we used 2 commercially available plasmids containing either SOX5 or NR4A3 3&#8242;UTR sequence inserted downstream of the firefly luciferase reporter gene, and containing in the same plasmid renilla luciferase gene for normalization. The representative results of 3&#8217; UTR luciferase activity down-regulation is presented in Figure 3.
<img alt="Figure 1" src="/files/ftp_upload/52410/52410fig1.jpg" /> 
Figure 1: Differentiation of hNSCs on 3D scaffolds, and measurement of axon processes outgrowth. (A) Following standard ICC staining for &#946;3-tubulin (green) and nuclei counterstained with Hoechst (blue) representative microphotographs were acquired and measurement of axon processes were performed with the aid of an image analysis software. (B) Diagrams reporting the measurement of axon process outgrowth performed in 2D and 3D differentiated hNSCs for 1 (1W) or 3 weeks (3W). <a href="https://www.jove.com/files/ftp_upload/52410/52410fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52410/52410fig2.jpg" /> 
Figure 2: Schematic workflow of miRNA profiling in differentiated hNSCs. Total RNA,including miRNAs, was extracted from either differentiated hNSCs on 3D scaffolds and standard 2D cultures, or undifferentiated control. 250 ng of total RNA were retro-transcribed with a method that facilitates the selective conversion of mature miRNAs into cDNA suitable for miRNA quantification with human cell differentiation and development miScript miRNA PCR array. The geo-mean of SNORD61, SNORD68, SNORD72, SNORD95, SNORD96A, and RNU6-2 was used for data analysis based on the 2-&#916;&#916;ct method. Significant changes were defined as +/- 1.5 fold up and down regulation to a statistically significant extent. Data were analyzed using web-based software available at Results are expressed as a clustergram. Modified from Figure 319. <a href="https://www.jove.com/files/ftp_upload/52410/52410fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52410/52410fig3.jpg" /> 
Figure 3: Validation of miRNA target predicted mRNA using a dual luciferase report assay. (A) Representative diagram of a dual luciferase reporter plasmid used as a tool to validate 3&#8217;UTR sequence as a direct target of a miRNA. (B) Representative micrograph showing transfection efficiency. (C)&#160;Signals, expressed as relative luciferase activity, from human 3&#39;UTR reporters ofSOX5 and NR4A3 genes were significantly knocked down in the presence of hsa-miR-96, and hsa-miR-7 and 17 miRNA mimics respectively. Modified from Figure 519. <a href="https://www.jove.com/files/ftp_upload/52410/52410fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td colspan="2">DIANALAB</td>
			<td>PicTar</td>
			<td>TargetScan</td>
		</tr>
		<tr>
			<td>miRNA</td>
			<td>Target gene</td>
			<td>Target sites/genes found</td>
			<td>Precision</td>
			<td>Score</td>
			<td>Aggregate PCT</td>
		</tr>
		<tr>
			<td>hsa-miR-96</td>
			<td>SOX5</td>
			<td>1214/763</td>
			<td>1</td>
			<td>6.74</td>
			<td>0.94</td>
		</tr>
		<tr>
			<td>hsa-miR-183</td>
			<td>DUSP10</td>
			<td>302/190</td>
			<td>0.72</td>
			<td>4.75</td>
			<td>0.85</td>
		</tr>
		<tr>
			<td>hsa-miR-302a</td>
			<td>NR4A3</td>
			<td>863/473</td>
			<td>0.84</td>
			<td>3.05</td>
			<td>NF</td>
		</tr>
		<tr>
			<td>hsa-miR-182</td>
			<td>FOXN3</td>
			<td>1358/794</td>
			<td>0.94</td>
			<td>NF</td>
			<td>0.96</td>
		</tr>
		<tr>
			<td>hsa-miR-7</td>
			<td>NR4A3</td>
			<td>NF</td>
			<td>NF</td>
			<td>1.61</td>
			<td>0.17</td>
		</tr>
		<tr>
			<td>hsa-miR-7</td>
			<td>FOXN3</td>
			<td>399/136</td>
			<td>0.17</td>
			<td>NF</td>
			<td>0.88</td>
		</tr>
		<tr>
			<td>hsa-miR-20a</td>
			<td>NR4A3</td>
			<td>1650/841</td>
			<td>0.85</td>
			<td>5.87</td>
			<td>0.63</td>
		</tr>
		<tr>
			<td>hsa-miR-20b</td>
			<td>NR4A3</td>
			<td>1955/973</td>
			<td>0.9</td>
			<td>5.87</td>
			<td>0.63</td>
		</tr>
		<tr>
			<td>hsa-miR-17</td>
			<td>NR4A3</td>
			<td>1928/961</td>
			<td>0.9</td>
			<td>5.38</td>
			<td>0.63+0.37</td>
		</tr>
		<tr>
			<td>hsa-miR-20a</td>
			<td>EIF4G3</td>
			<td>1650/841</td>
			<td>0.97</td>
			<td>NF</td>
			<td>NF</td>
		</tr>
		<tr>
			<td>hsa-miR-20b</td>
			<td>EIF4G3</td>
			<td>1955/973</td>
			<td>0.94</td>
			<td>NF</td>
			<td>NF</td>
		</tr>
		<tr>
			<td>hsa-miR-17</td>
			<td>EIF4G3</td>
			<td>1928/961</td>
			<td>0.94</td>
			<td>NF</td>
			<td>NF</td>
		</tr>
	</tbody>
</table>
Table 1: List of miRNA target prediction mRNAs. Representative table of miRNAs, target predictive genes,and algorithms analysis (prediction/score/aggregate provided using Diana Lab, PicTar, and TargetScan respectively). Modified from Table 319.

Watch this Scientific Journal Video about Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes at JoVE.com

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

With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

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9.8K Views.  ReNeuron. With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

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

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. 
The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods1-4 and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

It is critical in neurobiology and neurovirology to have a reliable, replicable in vitro system that serves as a translational model for what occurs in vivo in human neurons. This protocol describes how to culture and differentiate SH-SY5Y human neuroblastoma cells into viable neurons for use in in vitro applications.

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and ...

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental neurobiology. Rather than use an animal model or genetically modified induced pluripotent cells, human neural stem cells provide an effective in vitro system to examine the effects of treatments, screen drugs, or examine individual differences. Here, we provide the detailed protocols for methods used to expand human fetal brain neural stem cells in culture with serum-free media, to differentiate them into various neuronal subtypes and astrocytes via different priming procedures, and to freeze and recover these cells. Furthermore, we describe a procedure of using human fetal brain neural stem cells to study Zika virus infection.

This article details the methods that are used to expand human fetal brain neural stem cells in culture, as well as how to differentiate them into various neuronal subtypes and astrocytes, with an emphasis on the use of neural stem cells to study Zika virus infection.

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...