Name: differentiation of a human neural stem cell line on three dimensional cultures, analysis of microrna and putative target genes
Uploaded: 2015-04-12T14:00:00.000+00:00
Duration: 10 min 48 s
Description: 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

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><tbody><tr><td>DMEM:F12</td><td>Invitrogen</td><td>21331-020</td><td>For RMM medium preparation</td></tr><tr><td>Human Albumin Solution&amp;nbsp;</td><td>Baxter health care limited&amp;nbsp;</td><td>1501052</td><td>For RMM medium used at a final concentration of&amp;nbsp; 0.03%</td></tr><tr><td>Transferrin, Human recombinant</td><td>Sigma</td><td>T8158</td><td>For RMM medium used at a final concentration of&amp;nbsp; 5 &amp;micro;g/ml</td></tr><tr><td>Putrescine DiHCl&amp;nbsp;</td><td>Sigma</td><td>P5780</td><td>For RMM medium used at a final concentration of&amp;nbsp; 16.2 &amp;micro;g/ml</td></tr><tr><td>Insulin, Human recombinant&amp;nbsp;&amp;nbsp;</td><td>Sigma</td><td>I9278</td><td>For RMM medium used at a final concentration of&amp;nbsp; 5 &amp;micro;g/ml</td></tr><tr><td>Progesterone&amp;nbsp;</td><td>Sigma</td><td>P6149</td><td>For RMM medium used at a final concentration of&amp;nbsp; 60 ng/ml</td></tr><tr><td>L-Glutamine&amp;nbsp;</td><td>Invitrogen</td><td>25030-24</td><td>For RMM medium used at a final concentration of&amp;nbsp; 2 mM</td></tr><tr><td>Sodium Selenite&amp;nbsp;&amp;nbsp;</td><td>Sigma</td><td>S9133</td><td>For RMM medium used at a final concentration of 40 ng/ml</td></tr><tr><td>bFGF&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</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><td>EGF&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</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><td>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><td>Laminin</td><td>AMS Biotech</td><td>3400-010-10</td><td></td></tr><tr><td>TrypZean/EDTA</td><td>Lonza&amp;nbsp;</td><td>BE02-034E</td><td></td></tr><tr><td>Water for irrigation</td><td>Baxter Healthcare</td><td>UKF7114</td><td></td></tr><tr><td>Defined Trypsin Inhibitor (DTI) (store -20&amp;deg;C)</td><td>Invitrogen</td><td>R007-100</td><td></td></tr><tr><td>Alvetex 12 well plate&amp;nbsp;</td><td>Reinnervate Ltd</td><td>AVP002</td><td></td></tr><tr><td>Ethanol&amp;nbsp;</td><td>Merck&amp;nbsp;</td><td>1.00986.1000</td><td></td></tr><tr><td>&amp;beta;III tubulin antibody</td><td>Sigma</td><td>T8660</td><td></td></tr><tr><td>Hoechst</td><td>Sigma</td><td>B2261</td><td></td></tr><tr><td>miRNeasy mini kit</td><td>Qiagen&amp;nbsp;</td><td>217004</td><td></td></tr><tr><td>RNase free DNase&amp;nbsp;</td><td>Qiagen&amp;nbsp;</td><td>79254</td><td></td></tr><tr><td>Chloroform</td><td>Sigma</td><td>C7559-5VL</td><td></td></tr><tr><td>miScript II RT Kit&amp;nbsp;</td><td>Qiagen&amp;nbsp;</td><td>218160</td><td></td></tr><tr><td>SYBR green PCR kit</td><td>Qiagen&amp;nbsp;</td><td>218073</td><td></td></tr><tr><td>Cell Development &amp;amp; Differentiation miScript miRNA PCR Array</td><td>Qiagen/ SABiosciences</td><td>MIHS-103Z</td><td></td></tr><tr><td>Lightcycler 480 white 96 plate</td><td>Roche</td><td>&amp;nbsp;4729692001</td><td></td></tr><tr><td>Lightcycler 480&amp;nbsp; sealing foil</td><td>Roche</td><td>&amp;nbsp;4729757001</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen</td><td>11668-027</td><td></td></tr><tr><td>Vector NR4A3 miRNA 3&#039; UTR target clones&amp;nbsp;</td><td>GeneCopeia</td><td>HmiT019538-MT0</td><td></td></tr><tr><td>Vector SOX5 miRNA 3&#039; UTR target clones&amp;nbsp;</td><td>GeneCopeia</td><td>HmiT017632-MT01</td><td></td></tr><tr><td>Allstars negative control siRNA AF 488 (Qiagen).</td><td>Qiagen&amp;nbsp;</td><td>1027284</td><td>MiRNA transfection control</td></tr><tr><td>Luc-Pair miR Luciferase Assay</td><td>GeneCopeia</td><td>LPFR-M010</td><td></td></tr><tr><td>Lightcycler 480&amp;nbsp;</td><td>Roche</td><td></td><td></td></tr><tr><td>GloMax 96 microplate luminometer&amp;nbsp;</td><td>Promega</td><td></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 ...

differentiation-human-neural-stem-cell-line-on-three-dimensional

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

9.9K 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

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Bioengineering

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 ...

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...

Co-Culturing Microglia and Cortical Neurons Differentiated from Human Induced Pluripotent Stem Cells

The ability to generate microglia from human induced pluripotent stem cells (iPSCs) provides new tools and avenues for investigating the role of microglia in health and disease. Furthermore, iPSC-derived microglia can be maintained in co-culture with iPSC-derived cortical neurons, which enable investigations of microglia-neuron interactions that are hypothesized to be dysregulated in a number of neuropsychiatric disorders. Human iPSCs were differentiated to generate microglia using an adapted version of a protocol developed by the Fossati group, and the iPSC-derived microglia were validated with marker analysis and real-time PCR. Human microglia generated using this protocol were positive for the markers CD11C, IBA1, P2RY12, and TMEM119, and expressed the microglial-related genes AIF1, CX3CR1, ITGAM, ITGAX, P2RY12, and TMEM119. Human iPSC-derived cortical neurons that had been differentiated for 30 days were plated with microglia and maintained in co-culture until day 60, when experiments were undertaken. The density of dendritic spines in cortical neurons in co-culture with microglia was quantified under baseline conditions and in the presence of pro-inflammatory cytokines. In order to examine how microglia modulate neuronal function, calcium imaging experiments of the cortical neurons were undertaken using the calcium indicator Fluo-4 AM. Live calcium activity of cortical neurons was obtained using a confocal microscope, and fluorescence intensity was quantified using ImageJ. This report describes how co-culturing human iPSC-derived microglia and cortical neurons provide new approaches to interrogate the effects of microglia on cortical neurons.

This protocol describes a methodology to differentiate microglia from human iPSCs and maintain them in co-culture with iPSC-derived cortical neurons in order to study mechanistic underpinnings of neuroimmune interactions using human neurons and microglia.

The ability to generate microglia from human induced pluripotent stem cells (iPSCs) provides new tools and avenues for investigating the role of microglia ...

Neuroscience

Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential (MMP), mitochondrial production of reactive oxygen species (ROS), and mitochondrial DNA (mtDNA) copy number are often features of these processes. This report details a novel flow cytometry-based approach to measure multiple mitochondrial parameters in different cell types, including human induced pluripotent stem cells (iPSCs) and iPSC-derived neural and glial cells. This flow-based strategy uses live cells to measure mitochondrial volume, MMP, and ROS levels, as well as fixed cells to estimate components of the mitochondrial respiratory chain (MRC) and mtDNA-associated proteins such as mitochondrial transcription factor A (TFAM). 
By co-staining with fluorescent reporters, including MitoTracker Green (MTG), tetramethylrhodamine ethyl ester (TMRE), and MitoSox Red, changes in mitochondrial volume, MMP, and mitochondrial ROS can be quantified and related to mitochondrial content. Double staining with antibodies against MRC complex subunits and translocase of outer mitochondrial membrane 20 (TOMM20) permits the assessment of MRC subunit expression. As the amount of TFAM is proportional to mtDNA copy number, the measurement of TFAM per TOMM20 gives an indirect measurement of mtDNA per mitochondrial volume. The entire protocol can be carried out within 2-3 h. Importantly, these protocols allow the measurement of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry.

This study reports a novel approach to measure multiple mitochondrial functional parameters based on flow cytometry and double staining with two fluorescent reporters or antibodies to detect changes in mitochondrial volume, mitochondrial membrane potential, reactive oxygen species level, mitochondrial respiratory chain composition, and mitochondrial DNA.

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential ...

Differentiating Human Neuronal Stem Cells into 3D Culture Using a Porous Scaffold

Source: Stevanato, L. et al., <a href="https://app.jove.com/v/52410">Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes.</a> J. Vis. Exp. (2015)
This video showcases the development and differentiation of human neural stem cells within a three-dimensional scaffold. A laminin coating on the scaffold promotes cell attachment, while a growth factor-free neural medium induces cell differentiation into neural progenitor cells, which extend their processes within a porous architecture in multiple directions, forming a three-dimensional culture

1. HNSC differentiation on Three-Dimensional (3D) Culture
NOTE: Isolate and derive human neural stem cells (hNSCs) from an ethically approved tissue ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Advanced Neuronal Models

Differentiating Non-Transgenic and Transgenic Human Pluripotent Stem Cells into Neural Progenitor Cells

Source: Cvetkovic, C., et al. <a href="https://app.jove.com/v/58034">Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells.</a> J. Vis. Exp. (2018).
This video demonstrates the generation of neural progenitor cells from non-transgenic and transgenic human pluripotent stem cells (hPSCs). hPSCs are differentiated into neural progenitor cells using a neuronal differentiation medium. While differentiation is induced in non-transgenic cells using small molecules, the transgenic hPSCs are differentiated by adding an antibiotic that induces the promoter for a neuronal differentiation-specific transcription factor.

Ethic approvals were obtained when performing the experiments.
1. Cell Culture and Reagent Preparation

	Prepare coated plates for cell culture.
	
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Differentiating Human Embryonic Stem Cells into Neural Progenitors

Plate the cells on a basement membrane matrix coated 6-well plate at a density of 2 x 105 cells per well in 2 milliliters of mTeSR1 with 10 micromolar ROCK inhibitor. After 24 hours, replace the culture medium with neural induction medium supplemented with 1 micromolar dorsomorphin and 5 micromolar SB431542. Change the medium every other day during the first four days of neural induction, then every day, until confluence is reached at day seven.