Funding for this research was provided by the US Army Medical Research and Materiel Command (Grant account no. W81XWH-11-1-0700) and the Stem Cell Research Fund. PAB and EMP were recipients of Ames Laboratory Summer Undergraduate Laboratory Internships (SULI).

1. Substrate Preparation for 96-well Plates
<ol>
	<li>Create a map of the 96-well plate outlining the different substrates and cell-types to be examined (Figure&#160;1).</li>
	<li>Obtain the stock solutions of different substrates [poly-L-lysine, fibronectin, collagen type I, laminin, and entactin-collagen IV-laminin (ECL)], a 96-well multiwell plate and prepare a work station in a sterile cell culture hood.</li>
	<li>Prepare individual substrates by diluting stock in sterile phosphate buffered saline (PBS) to a final concentration of 5 &#181;g/ml (this concentration was previously determined based on a substrate concentration-dependent assay for growth and proliferation of cells). Mix using a vortex before pouring into a sterile reservoir.</li>
	<li>Add 100 &#181;l of substrate solution into each well according to the 96-well map (Figure 1) (a 12- or 8-channel micropipette is convenient for micropipetting into a 96-well plate). Seal the lid to the 96-well plate using a strip of Parafilm and store overnight at 4 &#176;C.</li>
</ol>
2. Cell Plating and Time-lapse Imaging
NOTE: Mouse MSCs were isolated from the bone marrow of adult C57BL/6 mice and maintained as an adherent cell line. MSCs were infected using lentiviral vectors to engineer them to secrete brain-derived neurotrophic factor (BDNF; human cDNA) and glial cell-derived neurotrophic factor (GDNF; human cDNA) using lentiviral vector&#8217;s encoding BDNF (LV-BDNF; CMV-BDNF-IRES-GFP), GDNF (LV-GDNF; CMV-GDNF-IRES-GFP), and green fluorescent protein (GFP, LV-GFP; CMV-GFP). 
NOTE: Culture media for mouse mesenchymal stem cells (MSCs) is Iscove&#39;s Modified Dulbecco&#39;s Medium containing 10% hybridoma-qualified fetal bovine serum, 10% equine serum, 2 mM L-glutamine, and 10,000 U/ml penicillin, 10 mg/ml streptomycin. The five different types of mouse MSCs (MSCs, GFP-MSCs, BDNF-GFP-MSCs, GDNF-GFP-MSCs and BDNF/GDNF-GFP-MSCs) were plated at about 30% confluence in T75 cell culture flasks.
<ol>
	<li>Cell Plating
	<ol>
		<li>On the following day, remove the substrate solutions by aspiration and rinse each well with approximately 200 &#181;l of sterile PBS, twice. Add cell culture media (200 &#181;l/well) to each well after the final PBS rinse. Place the 96-well plate into a cell culture incubator set at 37 &#176;C and 5% CO2 for equilibration.</li>
		<li>While the 96-well plate is equilibrating in the incubator, harvest the cells (MSCs in T75 flasks should be approximately 70% confluent at time of harvesting/plating) by collecting the growth media (referred to as conditioned media) from the T75 flask and storing in a 15 ml conical tube under sterile conditions (this conditioned media will be used in step 2.1.4 below).</li>
		<li>Add 8 ml of sterile PBS to the flask and gently swirl and then pipette off the PBS and add 1 ml of 0.05% trypsin and 0.01% EDTA solution to detach the cells from the culture surface of the T75 flask. Monitor cell detachment by viewing the flask using an inverted microscope equipped with phase contrast optics.</li>
		<li>When the cells have detached, immediately add 8 ml of the conditioned media (collected in step 2.1.2) to the flask. Collect the cell suspension and transfer to a 15 ml conical centrifuge tube and centrifuge for 4 min at 450 x g to pellet the cells.</li>
		<li>Remove the supernatant and resuspend the cell pellet in 200 &#181;l of fresh and warmed (37 &#176;C) cell culture media.</li>
		<li>Determine the number of cells in the cell suspension by performing a trypan blue viable cell count using a hemocytometer. Plate the cells at a density of approximately 300 cells/well into the appropriate wells of the 96-well plate.</li>
		<li>Repeat these steps for each population of cells.</li>
	</ol>
	</li>
	<li>Time-lapse Imaging
	<ol>
		<li>Once all of the MSCs have been plated, place the 96-well plate into an incubator for 2 hr to allow the MSCs to attach to the substrate.</li>
		<li>Start the HCS system and wait for 2 hr for the system to equilibrate. Set the environmental controller to 37 &#176;C and connect a mixed gas cylinder containing 5% CO2 in air to the HCS system environmental chamber supplying a constant air source.</li>
		<li>Remove the 96-well plate from the incubator following the 2 hr equilibration period and place directly into the cell growth chamber of the HCS system. Allow 30 min for equilibration to account for any heat-related expansion of the plate and then start the image acquisition and analysis software to configure the plate settings.</li>
		<li>Select the 20X objective for imaging. Select two wells per condition [i.e., GFP-MSCs on Fibronectin etc. (6 substrates x 5 MSC subtypes for a total of 30 conditions x 2 replicates = 60 wells in total)] for setting up time-lapse imaging. Choose two sites for imaging within each well.</li>
		<li>Choose the correct light wavelengths for imaging. 
		NOTE: Two different wavelengths (Phase contrast and GFP fluorescence) were selected for time-lapse imaging.</li>
		<li>Focus on the well bottom using laser autofocus and take test images for multiple sites and multiple wells to find an optimized focal plane.</li>
		<li>Once the focus has been established, begin capturing images every 5 min for 48 hr for all 60 wells (120 sites).</li>
		<li>Feed the cells every 24 hr by removing the 96-well plate from the HCS system. Remove 75 &#181;l of media from each well and add 100 &#181;l of fresh media to each well (equilibrate this fresh media at 37 &#176;C and 5% CO2).</li>
		<li>At the end of the time-lapse experiment, remove the 96-well plate from the HCS system. Under sterile conditions, collect the conditioned media samples from each well and transfer these samples to another 96-well plate. 
		NOTE: These samples can be used for further analysis by performing ELISA for neurotrophic factors.</li>
		<li>Prepare the 96-well plate with cultured MSCs for additional assays such as Ki67 cell proliferation assay or propidium iodide live/dead staining assay (see details below).</li>
		<li>Perform time-lapse imaging analysis for cell migration/cell tracking as described in section 5 below.</li>
	</ol>
	</li>
</ol>
3. Ki67 Cell Proliferation and Propidium Iodide Live/Dead Assay
<ol>
	<li>Ki67 Cell Proliferation Assay (Immunocytochemistry)
	<ol>
		<li>Rinse the cell cultures with 0.1 M phosphate (PO4) buffer for one minute two times. Fix the culture with 4% paraformaldehyde (PFA) for 20 min at room temperature. Remove the PFA and rinse the wells with PBS for seven minutes three times.</li>
		<li>After the final rinse, add 100 &#181;l of blocker solution (phosphate buffer saline, 5% normal donkey serum, 0.4% bovine albumin serum, and 0.2% Triton X-100) to each well and incubate at room temperature for 1 hr. Prepare the primary antibody, rabbit anti-Ki67, by diluting in blocker solution at a working ratio of 1:200.</li>
		<li>Remove the blocker solution and apply 100 &#181;l of the primary antibody solution to each well. Cover the 96-well plate and incubate the samples at 4 &#176;C overnight.</li>
		<li>On the following day, remove the antibody solution and rinse with PBS for 7 min, 3 times.</li>
		<li>Prepare the secondary antibody, donkey anti-rabbit Cy3 in blocking solution at a working ratio of 1:500. Add DAPI nuclear stain to the secondary antibody solution at a dilution of 1:100. After the removal of the last PBS rinse, apply 100 &#181;l of the secondary antibody/DAPI solution to each well. Incubate at room temperature in the dark for 90 min.</li>
		<li>Remove the secondary antibody/DAPI solution and rinse each well with PBS for 7 min, 3 times. Cover the 96-well plate and store at 4 &#176;C until imaging.</li>
	</ol>
	</li>
	<li>Propidium Iodide Live/Dead Assay
	<ol>
		<li>Measure cell death by propidium iodide (PI) exclusion assay as described below.</li>
		<li>Prepare the propidium iodide stain solution at a concentration of 1.5 &#181;M in culture media.</li>
		<li>Add 100 &#181;l of 70% ethanol to one well of the MSCs for 2 min with the intention of killing those cells. This well serves as a positive control for the PI stain. The majority of the MSCs will be PI-stained indicating cell death. Remove the ethanol solution.</li>
		<li>Add 100 &#181;l of propidium iodide stain solution to each well and incubate for 20 min at 37 &#176;C in a 5% CO2 incubator.</li>
		<li>Rinse the cells with 0.1 M phosphate buffer for 1 min, 2 times. Fix the cells with 4% PFA in 0.1 M P04 buffer for 20 min at room temperature. Remove the PFA and rinse with PBS three times for 7 min.</li>
		<li>Incubate with DAPI solution (1:50) diluted in blocker solution for 1 hr at room temperature. Rinse all wells with PBS for 7 min, 3 times.</li>
		<li>Remove the DAPI solution and rinse each well with PBS for 7 min, 3 times. Cover the 96-well plate and store at 4 &#176;C until imaging.</li>
	</ol>
	</li>
</ol>
4. Automated Imaging and Multiwavelength Scoring Analysis
<ol>
	<li>Load a 96-well plate (previously processed for Ki67 immunolabeling or propidium iodide-stained) into the HCS system and allow the plate to equilibrate for 20 min Open the HCS system image acquisition and analysis software.</li>
	<li>Choose the acquisition settings for the 10X objective using camera binning at 1 and a gain setting of 2. Find the Z-plane in which the cells reside by utilizing the Auto Exposure function and calculate the offset for each wavelength of interest. For this analysis, capture images for DAPI (W1), Cy3 (W2), and FITC (W3). Choose the maximum intensity level at which the negative control wells show no signal for image acquisition. Confirm this setting is appropriate for the positive wells.</li>
	<li>Acquire plate. Capture images and store them in the image acquisition and analysis software&#8217;s database.</li>
	<li>Once images have been acquired, open image acquisition and analysis offline software and review plate data from the images acquired above.</li>
	<li>Select the Multi-wavelength scoring analysis. Configure the minimum and maximum intensities for each wavelength. 
	NOTE: DAPI detection should mark the staining around each visible nucleus. Cy3 should detect the positive cells with Ki67 immunoreactivity (IR) and should not detect IR under the negative controls. For Cy3 Ki67 IR, the approximate minimum width was 7 &#181;m, approximate maximum width was 30 &#181;m, the intensity above the local background was 150 gray levels, and the minimum stained area was 50 &#181;m2.</li>
	<li>Run analysis for all positions. Export the data to view in spreadsheet.</li>
</ol>
5. Cell Tracking
<ol>
	<li>Open image acquisition and analysis program and click on &#8220;Review Plate Data [DB]&#8230;&#8221;, selecting the plate of interest.</li>
	<li>View the data as &#8220;Time vs. Well&#8221;.</li>
	<li>Choose one of the sites from the &#8220;Sites&#8221; section by left clicking on the desired selection. Select &#8220;Transmitted&#8230;&#8221; under the &#8220;Wavelengths:&#8221;. Right click on the target well in the 96-well plate template and click on &#8220;Load Images&#8221;.</li>
	<li>Track the cells by clicking on the &#8220;Apps&#8221; and then &#8220;Track Objects&#8221;.</li>
	<li>Use &#8220;Dynamic Data Exchange (DDE)&#8221; to choose the format to export the data, e.g., Microsoft Excel.</li>
	<li>Choose the tracking data to export, e.g., elapsed time, object number, distance, time interval, velocity, absolute angle, distance to origin, delta x and delta y.</li>
	<li>Tag each cell of interest with &#8220;Ctrl key + left click&#8221; on target cells.</li>
	<li>Track cells. If necessary, stop/change improper tracking of cells with the &#8220;Esc&#8221; key and then adjust the settings.</li>
	<li>After cell tracking is complete, save data with &#8220;Log Data&#8221;.</li>
</ol>

<ol><li>Thoenen, H., Sendtner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurotrophins%3A+from+enthusiastic+expectations+through+sobering+experiences+to+rational+therapeutic+approaches%5BTitle%5D)+AND+%22Nature+neuroscience%22%5BJournal%5D)">Neurotrophins: from enthusiastic expectations through sobering experiences to rational therapeutic approaches.</a> Nature neuroscience. 5 Suppl, 1046-1050 (2002).</li>
<li>Park, H., Poo, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurotrophin+regulation+of+neural+circuit+development+and+function%5BTitle%5D)+AND+%22Nature+reviews%22%5BJournal%5D)">Neurotrophin regulation of neural circuit development and function.</a> Nature reviews. 14, 7-23 (2013).</li>
<li>Lin, L. F., Doherty, D. H., Lile, J. D., Bektesh, S., Collins, F. GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aLin%2C+LF+%22Science+%28New+York%2C+N.Y.%29%22">Science (New York, N.Y.)</a>. 260, 1130-1132 (1993).</li>
<li>Azizi, S. A., Stokes, D., Augelli, B. J., DiGirolamo, C., Prockop, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engraftment+and+migration+of+human+bone+marrow+stromal+cells+implanted+in+the+brains+of+albino+rats--similarities+to+astrocyte+grafts%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats--similarities to astrocyte grafts.</a> Proceedings of the National Academy of Sciences of the United States of America. 95, 3908-3913 (1998).</li>
<li>Prockop, D. J., Gregory, C. A., Spees, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((One+strategy+for+cell+and+gene+therapy%3A+harnessing+the+power+of+adult+stem+cells+to+repair+tissues%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">One strategy for cell and gene therapy: harnessing the power of adult stem cells to repair tissues.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 Suppl 1, 11917-11923 (2003).</li>
<li>Akiyama, Y., Radtke, C., Honmou, O., Kocsis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Remyelination+of+the+spinal+cord+following+intravenous+delivery+of+bone+marrow+cells%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">Remyelination of the spinal cord following intravenous delivery of bone marrow cells.</a> Glia. 39, 229-236 (2002).</li>
<li>Hofstetter, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Marrow+stromal+cells+form+guiding+strands+in+the+injured+spinal+cord+and+promote+recovery%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Marrow stromal cells form guiding strands in the injured spinal cord and promote recovery.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 2199-2204 (2002).</li>
<li>Chopp, M., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Treatment+of+neural+injury+with+marrow+stromal+cells%5BTitle%5D)+AND+%22Lancet+neurology%22%5BJournal%5D)">Treatment of neural injury with marrow stromal cells.</a> Lancet neurology. 1, 92-100 (2002).</li>
<li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transplantation+of+marrow+stromal+cells+restores+cerebral+blood+flow+and+reduces+cerebral+atrophy+in+rats+with+traumatic+brain+injury%3A+in+vivo+MRI+study%5BTitle%5D)+AND+%22Journal+of+neurotrauma%22%5BJournal%5D)">Transplantation of marrow stromal cells restores cerebral blood flow and reduces cerebral atrophy in rats with traumatic brain injury: in vivo MRI study.</a> Journal of neurotrauma. 28, 535-545 (2011).</li>
<li>Jin, H. K., Carter, J. E., Huntley, G. W., Schuchman, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracerebral+transplantation+of+mesenchymal+stem+cells+into+acid+sphingomyelinase-deficient+mice+delays+the+onset+of+neurological+abnormalities+and+extends+their+life+span%5BTitle%5D)+AND+%22The+Journal+of+clinical+investigation%22%5BJournal%5D)">Intracerebral transplantation of mesenchymal stem cells into acid sphingomyelinase-deficient mice delays the onset of neurological abnormalities and extends their life span.</a> The Journal of clinical investigation. 109, 1183-1191 (2002).</li>
<li>Harper, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transplantation+of+BDNF-secreting+mesenchymal+stem+cells+provides+neuroprotection+in+chronically+hypertensive+rat+eyes%5BTitle%5D)+AND+%22Investigative+ophthalmology+%26+visual+science%22%5BJournal%5D)">Transplantation of BDNF-secreting mesenchymal stem cells provides neuroprotection in chronically hypertensive rat eyes.</a> Investigative ophthalmology & visual science. 52, 4506-4515 (2011).</li>
<li>Arnhold, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adenovirally+transduced+bone+marrow+stromal+cells+differentiate+into+pigment+epithelial+cells+and+induce+rescue+effects+in+RCS+rats%5BTitle%5D)+AND+%22Investigative+ophthalmology+%26+visual+science%22%5BJournal%5D)">Adenovirally transduced bone marrow stromal cells differentiate into pigment epithelial cells and induce rescue effects in RCS rats.</a> Investigative ophthalmology & visual science. 47, 4121-4129 (2006).</li>
<li>Inoue, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subretinal+transplantation+of+bone+marrow+mesenchymal+stem+cells+delays+retinal+degeneration+in+the+RCS+rat+model+of+retinal+degeneration%5BTitle%5D)+AND+%22Experimental+eye+research%22%5BJournal%5D)">Subretinal transplantation of bone marrow mesenchymal stem cells delays retinal degeneration in the RCS rat model of retinal degeneration.</a> Experimental eye research. 85, 234-241 (2007).</li>
<li>Xia, X., Wong, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Concise+review%3A+a+high-content+screening+approach+to+stem+cell+research+and+drug+discovery%5BTitle%5D)+AND+%22Stem+cells+%28Dayton%2C+Ohio%29%22%5BJournal%5D)">Concise review: a high-content screening approach to stem cell research and drug discovery.</a> Stem cells (Dayton, Ohio). 30, 1800-1807 (2012).</li>
<li>Scholzen, T., Gerdes, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Ki-67+protein%3A+from+the+known+and+the+unknown%5BTitle%5D)+AND+%22Journal+of+cellular+physiology%22%5BJournal%5D)">The Ki-67 protein: from the known and the unknown.</a> Journal of cellular physiology. 182, 311-322 (2000).</li>
<li>Kapuscinski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DAPI%3A+a+DNA-specific+fluorescent+probe%5BTitle%5D)+AND+%22Biotechnic+%26+histochemistry+%3A+official+publication+of+the+Biological+Stain+Commission%22%5BJournal%5D)">DAPI: a DNA-specific fluorescent probe.</a> Biotechnic & histochemistry : official publication of the Biological Stain Commission. 70, 220-233 (1995).</li>
<li>Jiang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pluripotency+of+mesenchymal+stem+cells+derived+from+adult+marrow%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Pluripotency of mesenchymal stem cells derived from adult marrow.</a> Nature. 418, 41-49 (2002).</li>
<li>Woodbury, D., Schwarz, E. J., Prockop, D. J., Black, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adult+rat+and+human+bone+marrow+stromal+cells+differentiate+into+neurons%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Research%22%5BJournal%5D)">Adult rat and human bone marrow stromal cells differentiate into neurons.</a> Journal of Neuroscience Research. 61, 364-370 (2000).</li>
<li>Huang, B., Tabata, Y., Gao, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stem+cells+as+therapeutic+agents+and+potential+targeted+gene+delivery+vehicle+for+brain+diseases%5BTitle%5D)+AND+%22Journal+of+controlled+release+%3A+official+journal+of+the+Controlled+Release+Society%22%5BJournal%5D)">Mesenchymal stem cells as therapeutic agents and potential targeted gene delivery vehicle for brain diseases.</a> Journal of controlled release : official journal of the Controlled Release Society. 162, 464-473 (2012).</li>
<li>Uccelli, A., Benvenuto, F., Laroni, A., Giunti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuroprotective+features+of+mesenchymal+stem+cells%5BTitle%5D)+AND+%22Best+Pract+Res+Clin+Haematol%22%5BJournal%5D)">Neuroprotective features of mesenchymal stem cells.</a> Best Pract Res Clin Haematol. 24, 59-64 (2011).</li>
<li>Forostyak, S., Jendelova, P., Sykova, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+mesenchymal+stromal+cells+in+spinal+cord+injury%2C+regenerative+medicine+and+possible+clinical+applications%5BTitle%5D)+AND+%22Biochimie%22%5BJournal%5D)">The role of mesenchymal stromal cells in spinal cord injury, regenerative medicine and possible clinical applications.</a> Biochimie. 95, 2257-2270 (2013).</li>
<li>Shi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+lentivirus-mediated+TH+and+GDNF+genetic+engineering+mesenchymal+stem+cells+on+Parkinson%27s+disease+rat+model%5BTitle%5D)+AND+%22Neurological+sciences+%3A+official+journal+of+the+Italian+Neurological+Society+and+of+the+Italian+Society+of+Clinical+Neurophysiology%22%5BJournal%5D)">The effect of lentivirus-mediated TH and GDNF genetic engineering mesenchymal stem cells on Parkinson's disease rat model.</a> Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology. 32, 41-51 (2011).</li>
<li>Harper, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brain-derived+neurotrophic+factor+released+from+engineered+mesenchymal+stem+cells+attenuates+glutamate-+and+hydrogen+peroxide-mediated+death+of+staurosporine-differentiated+RGC-5+cells%5BTitle%5D)+AND+%22Experimental+eye+research%22%5BJournal%5D)">Brain-derived neurotrophic factor released from engineered mesenchymal stem cells attenuates glutamate- and hydrogen peroxide-mediated death of staurosporine-differentiated RGC-5 cells.</a> Experimental eye research. 89, 538-548 (2009).</li>
<li>Harrill, J. A., Freudenrich, T. M., Machacek, D. W., Stice, S. L., Mundy, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+assessment+of+neurite+outgrowth+in+human+embryonic+stem+cell-derived+hN2+cells+using+automated+high-content+image+analysis%5BTitle%5D)+AND+%22Neurotoxicology%22%5BJournal%5D)">Quantitative assessment of neurite outgrowth in human embryonic stem cell-derived hN2 cells using automated high-content image analysis.</a> Neurotoxicology. 31, 277-290 (2010).</li>
</ol>

Publication fees for this video article were supported by Molecular Devices, LLC.

Adult mesenchymal stem cells (MSCs) are an attractive cell type for development of an experimental strategy combining a cellular and gene delivery based therapy. MSCs are multipotent, capable of differentiating into cells of mesodermal lineage, and display considerable plasticity, differentiating/transdifferentiating into neuronal and glial lineages with the appropriate induction paradigms17,18. Furthermore, MSCs have been transplanted and proven effective in preclinical studies for a number of disorders, including neurodegenerative conditions19. The therapeutic efficacy of MSCs is well known due to their beneficial anti-proliferative, anti-inflammatory and anti-apoptotic activities20. MSCs are also known to produce and secrete various neurotrophic and growth factors, which likely contributes to the neuroprotective qualities associated with na&#239;ve MSCs following transplantation at sites of injury or disease21. Importantly, MSCs can be genetically modified for sustained delivery of neurotrophic factors for combined cellular and gene therapy-based applications and have been used in a number of animal models of injury or disease to the CNS11,19,22.
In developing a combined cellular and gene therapy-based strategy, it is important that the health of the cells is carefully assessed prior to their extensive use for in vitro, and especially in vivo applications. As a proof of concept, we investigated multiple populations of engineered and control MSC lines in order to study the consequences of the genetic modifications on cell health and fitness using a high content screening (HCS) approach. In general, HCS refers to cellular image-based high throughput screening14. This screening approach permits a quantitative assessment of cellular phenotypes at multiple levels of spatial (cell to subcellular) and temporal (milliseconds to days) resolution across various experimental conditions. Using this approach we accessed possible differences in substrate preference on the following parameters: cell proliferation, expression of green fluorescent protein (GFP), cell death, and cell motility/migration. Experiments were designed in 96-well cell culture plate format. Within a single plate we routinely investigated possible substrate-related differences with respect to each parameter for the different MSC populations of lentiviral-transduced cells and compared the engineered MSCs with the original, non-transduced MSCs. This provided a means to directly compare the results for the different MSC subtypes with a battery of in vitro assays such as cell proliferation using Ki67 immunolabeling, live/dead cell viability assay using propidium iodide staining, and cell behavior by performing time-lapse digital imaging. As an extension of this HCS one can also perform ELISAs on conditioned media samples collected from individual wells to quantitatively determine secretion of neurotrophic factors. Conditioned media from different MSC subtypes may also be used in in vitro bioassays to determine biological activity of secreted factors11,23. This type of HCS platform may also be used for in vitro measurements of neurite outgrowth from primary neuronal cultures and neural stem cell lines24. Overall, our results demonstrated that the subpopulations of the genetically modified MSCs displayed similar behaviors in comparison to the non-modified MSCs. The influence of diverse culture substrates on cell growth and cell migration was not dramatically different between the MSC subtypes, as well as culture substrates. As such, the extracellular matrix substrates tested did not appear to play a critical role in modulating these aspects of cell behavior for these different engineered MSCs.
This study demonstrates the use of an HCS system for analyzing different aspects of cell behavior. However, it is not uncommon to encounter limitations associated with image analysis. On occasion, while analyzing the fluorescence images, it was somewhat difficult to determine the correct threshold value above which immunolabeled or stained cells would be counted as positively labeled/stained. Thus, to minimize subjective bias, the determination of threshold values was dependent upon a comparison with controls (negative controls for fluorescence imaging were carried out in parallel during all processing by the omission of the primary or secondary antibodies). Another limitation was encountered during the analysis of cell migration using time- lapse digital imaging. In some cases, the imaging software was not able to differentiate between random Brownian motion of a cell versus a cell actually migrating only a very short distance. Additional limitations were evident in situations where the analysis software was not able to distinguish the presence of multiple cells in very close proximity to one-another. To overcome this limitation required manual cell selection during the analysis rather than a fully automated analysis. Cell plating density can also result in skewing cell migration data between populations of cells that display greater preference to grow in clumps versus cells that grow in isolation from each other. These types of differences are in part likely a reflection of cell-substrate versus cell-cell preferences.
Using an HCS system to acquire images and perform data analysis provides an efficient and rapid means to assess multiple cell parameters. In addition, time-lapse digital videos for 30 different conditions (6 substrates and 5 different MSC subtypes) were routinely acquired for periods ranging from hours to days (48 hr) while using the environmental chamber. This data was subsequently used to calculate and determine differences in cell migration rates across various cell lines on different ECM molecules.
In this report we have highlighted the implementation of a high content screening platform to assess cell health and function. This type of analysis is useful for developing rational strategies for designing cell-types, as well as polymer substrates to facilitate directed cell growth and neural regeneration. This is an essential step towards application of stem cell-based delivery of therapeutic factors prior to extensive in vivo preclinical studies using cell transplantation strategies.

A major issue with implementing useful therapies for treatment of nervous system disorders is in developing effective methods that prevent further degeneration and also facilitate recovery of function. An innovative strategy is to genetically engineer stem cells ex vivo, for production of neuroprotective factors, prior to their transplantation. This combination of cell-based therapy, coupled with a type of gene therapy, provides a powerful method for the treatment of disease or injury-induced neuronal death in the nervous system.
Neurotrophic factors are essential for growth and survival of developing neurons as well as maintenance and plasticity of mature neurons. A number of studies have demonstrated significant roles of neurotrophic factors in promoting initial growth and differentiation of neurons in the central and peripheral nervous system (CNS and PNS) and they can also stimulate regeneration in vitro and in animal models of neural injury1. Brain-derived neurotrophic factor (BDNF) is highly expressed in the CNS and plays important roles in regulating neural development, synaptic plasticity and repair2. Glial cell line-derived neurotrophic factor (GDNF) promotes survival of many types of neurons including dopaminergic and motorneurons3. Thus, an important strategy for neural repair is to provide exogenous sources of neurotrophic factors to the injured or diseased regions of the nervous system.
Multipotent bone marrow-derived mesenchymal stem cells (MSCs) hold great potential for delivery of therapeutic proteins to treat the damaged or diseased nervous system. Transplantation of MSCs has attracted considerable attention in efforts to develop patient compatible cell-based therapies since they have a number of advantages including, 1) relative ease of isolation and maintenance, 2) multipotential capacity, 3) little ethical concerns, 4) ability to survive and migrate following transplantation and 5) potential for autologous transplantation4,5. Promising results have been reported with use of na&#239;ve and genetically engineered MSCs in animal models for a number of different neurodegenerative conditions, including spinal cord injury6,7, stroke8,9, myelin deficiency10, and retinal degeneration11-13. Coupling cell transplantation with delivery of neurotrophic factors from genetically engineered stem cells is a novel and important neural repair strategy.
An essential step in developing cell-based therapeutic factor delivery systems is to determine the normal health of the engineered cells. As such, the principal purpose of this study was to evaluate general growth parameters of genetically engineered adult stem cells. An important approach to rapidly assess multiple cell parameters is to employ cellular image-based high-through screening (HTS), often referred to as high content screening (HCS) procedures14. This technology allows automated image acquisition and analysis and this approach is particularly well suited for stem cell research applications. In this project we developed a profiling platform that allows for the rapid characterization and optimization of cell substrate preferences and cellular functions with genetically engineered adult stem cells employing a HCS system.

<table><tbody><tr><td>96 well plates</td><td>Greiner Bio One</td><td>655090</td><td>96 well plates selected for use in ImageXpress</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich (St. Louis, MO)</td><td>A9647</td><td></td></tr><tr><td>Rabbit anti-Ki67 antibody</td><td>Abcam (Cambridge, MA)</td><td>Ab16667</td><td>1:200 dilution</td></tr><tr><td>Collagen type I</td><td>Sigma-Aldrich (St. Louis, MO)</td><td>C7661</td><td>Collagen from rat tail</td></tr><tr><td>DAPI</td><td>Invitrogen (Carlsbad, CA)</td><td>D3571</td><td></td></tr><tr><td>Donkey anti-Rabbit Cy3</td><td>Jackson Immuno Res Lab (West Grove, PA)&amp;nbsp;</td><td>711-165-152</td><td>1:500 dilution</td></tr><tr><td>ECL(Entactin-Collagen-Laminin)</td><td>Millipore/Chemicon (Temecula, CA)</td><td>08-110</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Hyclone (Logan, UT)</td><td>SH30071.03</td><td></td></tr><tr><td>Equine serum</td><td>Hyclone (Logan, UT)</td><td>SH3007403</td><td></td></tr><tr><td>Ethanol</td><td>Chemistry Store (Ames, IA)</td><td>12003510</td><td>100%, 200 proof</td></tr><tr><td>Fibronectin</td><td>Fisher Scientific (Hampton, NH)</td><td>CB-40008</td><td></td></tr><tr><td>Iscove&amp;rsquo;s Modified Dulbeccos Medium (IMDM)</td><td>Hyclone (Logan, UT)</td><td>SH30396.03</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific (Hampton, NH)</td><td>P285</td><td>For PO&lt;sub&gt;4&lt;/sub&gt; buffer</td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific (Hampton, NH)</td><td>P288</td><td>For PO&lt;sub&gt;4&lt;/sub&gt; buffer</td></tr><tr><td>L-Glutamine</td><td>Gibco/Invitrogen (Grand Island, NY)</td><td>25030-081</td><td></td></tr><tr><td>Laminine (mouse)</td><td>Trevigen (Gaithersburg, MD)</td><td>3400-010-01</td><td></td></tr><tr><td>Mouse MSCs of adult C57BL/6 mice</td><td>Tulane Cent for Gene Therapy (New Orleans, LA)</td><td></td><td>Isolated from bone marrow</td></tr><tr><td>Genetically modified MSCs (GFP, BDNF, GDNF, BDNF/GDNF)</td><td>These cells were obtained from our previous study : Ye et. al. (in preparation)</td><td></td><td></td></tr><tr><td>Normal donkey serum (NDS)</td><td>Jackson Immuno Res Lab (West Grove, PA)&amp;nbsp;</td><td>017-000-121</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Fisher Scientific (Hampton, NH)</td><td>O4042</td><td>4% PFA in 0.1 M PO&lt;sub&gt;4&lt;/sub&gt; buffer</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich (St. Louis, MO)</td><td>P0781</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich (St. Louis, MO)</td><td>P4417</td><td></td></tr><tr><td>Poly-L-lysine</td><td>Sigma-Aldrich (St. Louis, MO)</td><td>P4707</td><td></td></tr><tr><td>Propidium iodide (PI)</td><td>Invitrogen (Carlsbad, CA)</td><td>P1304MP</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich (St. Louis, MO)</td><td>X100</td><td></td></tr><tr><td>Trypsin 0.05% (EDTA 1x)</td><td>Invitrogen (Carlsbad, CA)</td><td>25300-054</td><td></td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium</td><td>Invitrogen (Carlsbad, CA)</td><td>12440&amp;ndash;046</td><td></td></tr><tr><td>Hybridoma-qualified FBS</td><td>Hyclone (Logan, UT)</td><td>SH30396.03</td><td></td></tr><tr><td>Equine serum</td><td>Hyclone (Logan, UT)</td><td>SH3007403</td><td></td></tr><tr><td>ImageXpress Micro</td><td>Molecular devices (Sunnyvale, CA)</td><td>ImageXpress micro</td><td>High content screening system</td></tr><tr><td>MetaXpress 4.0</td><td>Molecular devices (Sunnyvale, CA)</td><td>MetaXpress 4.0</td><td>Image acquisition and analysis software</td></tr></tbody></table>

high throughput characterization of adult stem cells engineered for delivery of therapeutic factors for neuroprotective strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

MSC growth parameters were examined by culturing the different populations of MSCs on different substrates. The five different populations of MSC subtypes (MSCs, GFP-MSCs, BDNF-GFP-MSCs, GDNF-GFP-MSCs, and BDNF/GDNF-GFP-MSCs) were plated into 96-well tissue culture plates pre-coated with the different substrates as illustrated in Figure 1. After four days of culturing, the plates were fixed and immunolabeled and/or stained with the appropriate reagents and then examined using the HCS system and analyses conducted with the image acquisition and analysis software program.
<img alt="Figure 1" src="/files/ftp_upload/52242/52242fig1highres.jpg" width="650" /> 
Figure 1: 96-well plate template for experimental design. 96-well plates were coated with various substrates and wells were seeded with engineered stem cells as shown in the template. As an example, only wells in rows B-F were used in this experiment. Rows A, G and H were left empty. Abbreviations &#8211; MSCs: mesenchymal stem cells; GFP-MSCs: green fluorescent protein-expressing MSCs; BDNF-GFP-MSCs: brain derived neurotrophic factor-GFP-expressing MSCs; GDNF-GFP-MSCs; Glial cell-derived neurotrophic factor-GFP-expressing MSCs; BDNF/GDNF-GFP-MSCs; BDNF and GDNF- GFP-expressing MSCs; ECL: Entactin-Collagen IV-Laminin).
Anti-Ki67 immunolabeling, followed by DAPI counterstaining, was used to evaluate whether the different substrates influenced proliferation of the different populations of engineered MSCs (Figure 2A). Expression of the Ki67 antigen occurs preferentially during late G1, S, G2 and M phases of the cell cycle, and is not detected in cells in the resting phase (G0), and therefore is useful as a cellular marker for proliferation15. 4&#39;,6-diamidino-2-phenylindole (DAPI) is a commonly used nuclear and chromosome counterstain that emits blue fluorescence upon binding to AT regions of DNA16. The total number of cells in a field can be determined by counting the number of DAPI stained nuclei. As illustrated in Figure 2B, although there was variation in the percentages of proliferating MSCs, all substrates nevertheless supported considerable cell proliferation for each of the MSC subtypes.
<img alt="Figure 2" src="/files/ftp_upload/52242/52242fig2highres.jpg" width="700" /> 
Figure 2: Ki67 cell proliferation assay. (A) Merged, double-fluorescent image of Ki67 immunolabeling (red) and DAPI (blue) nuclear staining. Many of the MSCs were immunolabeled with the Ki67 antibody (red). Scale bar = 50 &#181;m. (B) Bar graph illustrating the percentages of Ki67 immunolabeled MSC subtypes grown on polystyrene (PS), poly-L-lysine (PLL), fibronectin, collagen type I, laminin, or entactin-collagen IV-laminin (ECL) substrates for 5 days in vitro (DIV). N = one experiment. Each bar represents averaged pooled data from 8 imaged sites from 2 wells for each condition. <a href="https://www.jove.com/files/ftp_upload/52242/52242fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Propidium iodide (PI) staining was used to evaluate whether different substrates influenced cell survival (Figure 3). Propidium iodide is a commonly used red-fluorescent nuclear and chromosome counterstain. Propidium iodide is membrane impermeant and generally excluded from viable cells, and thus is useful to detect dead cells in a population. The proportion of dead cells within a given condition can be determined when combined with a general nuclear label such as DAPI to identify all cells within a field. The percentage of PI-positive cells was low on all substrates examined (Figure 3). As a positive control for the PI reagent, a few wells containing MSCs were incubated in 70% ethanol, a condition known to kill most cells, resulting in a high percentage of PI-labeled cells as illustrated in Figure 3B and 3C (ethanol treated positive control).
<img alt="Figure 3" src="/files/ftp_upload/52242/52242fig3highres.jpg" width="700" /> 
Figure 3: Propidium iodide cell death assay. (A) Merged, double fluorescent image for propidium iodide (red) and DAPI (blue) staining. Although the nuclei of all of the viable cells were stained with DAPI (blue), no propidium iodide staining was detected in the MSCs. (B) Virtually all MSCs were stained with propidium iodide following exposure to 70% ethanol. Scale bars in A and B = 100 &#181;m. (C) Bar graph illustrating the percentages of propidium iodide (PI) stained MSC subtypes grown on polystyrene (PS), poly-L-lysine (PLL), fibronectin, collagen type I, laminin, or entactin-collagen IV-laminin (ECL) substrates for 5 days in vitro (DIV). Ethanol Control: This condition served as a positive control for the PI staining reagent. Most cells subjected to PI stain following ethanol treatment are dead and thus positively stained for the PI reagent. N = one experiment. Each bar represents averaged pooled data from 8 imaged sites from 2 wells for each condition. <a href="https://www.jove.com/files/ftp_upload/52242/52242fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To investigate the possible influence of different substrates on the behavior of engineered MSCs, cell migration was analyzed using time-lapse digital microscopy and the transmitted light/environmental chamber system on the HCS system (see Supplemental Video 1). Multiple sites/well were time-lapse imaged and used to calculate cell migration rates for the different subpopulations of MSCs growing on the different substrates using the image acquisition and analysis software program. In general, as shown in Figure 4C, all subtypes of MSCs showed the fastest migration rate on the extracellular matrix-coated surfaces (Fibronectin, Collagen, Laminin and ECL) and the slowest on non-coated polystyrene surfaces.
<img alt="Figure 4" src="/files/ftp_upload/52242/52242fig4highres.jpg" width="700" /> 
Figure 4: Cell tracking and migration. MSCs tracked with image acquisition and analysis software. Overlayed images of transmitted light and fluorescence images from (A) the start of time-lapse imaging and (B) at 29 hr later at the end of the time-lapse imaging session (see Supplemental Video 1). Cell migration tracks are indicated by the colored lines. Scale bar: 50 &#181;m. (C) Bar graph illustrating the average migration rates (expressed as &#181;m/hr) for MSC subtypes grown on polystyrene (PS), poly-L-lysine (PLL), fibronectin, collagen type I, laminin, or entactin-collagen IV-laminin (ECL) substrates for 2 days in vitro (DIV). N = one experiment. Each bar represents the average of at least 10 imaged cells from 2 wells for each condition. <a href="https://www.jove.com/files/ftp_upload/52242/52242fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Taken together, these results provide preliminary evidence that these subpopulations of genetically engineered MSCs display similar growth properties. These results provide compelling evidence that the lentiviral mediated genetic modifications of these MSCs induced no dramatic detectable deleterious effects on the growth parameters investigated using this screening platform.
Supplemental Video 1. Time-lapse digital video of MSC tracking using the image acquisition and analysis software program. The migration path&#8217;s for two MSCs [indicated as 1 (green tracking line) and 2 (Blue tracking line)] are illustrated. Video captured during a 29 hr period. Images were captured every 5 min. Fluorescence images of GFP-expressing MSCs was used for time-lapse imaging in preparing the video. Calibration bar = 50 &#181;m. This type of analysis is useful for investigating cell behaviors, including cell migration and cell division.

Watch this Scientific Journal Video about High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies at JoVE.com

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

high-throughput-characterization-adult-stem-cells-engineered-for

Research

JoVE Journal

Medicine

10.8K Views.  Iowa State University. This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

Video: High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Developmental Biology

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Neuroscience

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven therapies for TBI have been developed. This paper presents a novel method for pre-clinical neurotrauma research intended to complement existing pre-clinical models. It introduces human pathophysiology through the use of human induced pluripotent stem cell-derived neurons (hiPSCNs). It achieves loading pulse duration similar to the loading durations of clinical closed head impact injury. It employs a 96-well format that facilitates high throughput experiments and makes efficient use of expensive cells and culture reagents. Silicone membranes are first treated to remove neurotoxic uncured polymer and then bonded to commercial 96-well plate bodies to create stretchable 96-well plates. A custom-built device is used to indent some or all of the well bottoms from beneath, inducing equibiaxial mechanical strain that mechanically injures cells in culture in the wells. The relationship between indentation depth and mechanical strain is determined empirically using high speed videography of well bottoms during indentation. Cells, including hiPSCNs, can be cultured on these silicone membranes using modified versions of conventional cell culture protocols. Fluorescent microscopic images of cell cultures are acquired and analyzed after injury in a semi-automated fashion to quantify the level of injury in each well. The model presented is optimized for hiPSCNs but could in theory be applied to other cell types.

Here we present a method for a human in vitro model of stretch injury in a 96-well format on a timescale relevant to impact trauma. This includes methods for fabricating stretchable plates, quantifying the mechanical insult, culturing and injuring cells, imaging, and high content analysis to quantify injury.

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Biology

GM-Free Generation of Blood-Derived Neuronal Cells

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other therapeutic strategies, there is currently no cure available for the injured or diseased brain. Cell replacement appears as a promising therapeutic strategy for neurodegenerative conditions. To this day, neural stem cells (NSCs) have been successfully generated from fetal tissues, human embryonic cells (ES) or induced pluripotent stem cells (iPSC). A process of dedifferentiation was initiated by activation of the novel human GPI-linked glycoprotein, which leads to generation of pluripotent stem cells. These blood-derived pluripotent stem cells (BD-PSCs) differentiate in vitro into cells with a neural phenotype as shown by brightfield and immunofluorescence microscopy. Ultrastructural analysis of these cells by means of electron microscopy confirms their primitive structure as well as neuronal-like morphology and subcellular characteristics.

We present a genetically modified-free (GM-free) method to obtain cells with a neuronal phenotype from reprogrammed peripheral blood cells. Activation of a signaling pathway linked to novel human GPI-linked protein reveals an efficient GM-free method for obtaining human pluripotent stem cells.

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other ...

High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance between in vitro simplification and recreating the complex in vivo environment, along with the main aim, shared by all screening strategies, of obtaining robust and reliable data, highly predictive for in vivo translation.
In the field of demyelinating diseases, the majority of drug screening strategies are based on immortalized cell lines or pure cultures of isolated primary oligodendrocyte precursor cells (OPCs) from newborn animals, leading to strong biases due to the lack of age-related differences and of any real pathological condition or complexity.
Here we show the setup of an in vitro system aimed at modeling the physiological differentiation/maturation of neural stem cell (NSC)-derived OPCs, easily manipulated to mimic pathological conditions typical of demyelinating diseases. Moreover, the method includes isolation from fetal and adult brains, giving a system which dynamically differentiates from OPCs to mature oligodendrocytes (OLs) in a spontaneous co-culture which also includes astrocytes. This model physiologically resembles the thyroid hormone-mediated myelination and myelin repair process, allowing the addition of pathological interferents which model disease mechanisms. We show how to mimic the two main components of demyelinating diseases (i.e., hypoxia/ischemia and inflammation), recreating their effect on developmental myelination and adult myelin repair and taking all the cell components of the system into account throughout, while focusing on differentiating OPCs.
This spontaneous mixed model, coupled with cell-based high-content screening technologies, allows the development of a robust and reliable drug screening system for therapeutic strategies aimed at combating the pathological processes involved in demyelination and at inducing remyelination.

We describe the production of mixed cultures of astrocytes and oligodendrocyte precursor cells derived from fetal or adult neural stem cells differentiating into mature oligodendrocytes, and in vitro modeling of noxious stimuli. The coupling with a cell-based high-content screening technique builds a reliable and robust drug screening system.

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance ...

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...

Efficient Nucleofection of NSCs Isolated from the Murine Subventricular Zone

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

Author Spotlight: Improved Nucleofection for High-Efficiency Gene Delivery in Murine Subventricular Zone-Derived Neural Stem Cell Cultures

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...