Kristen Brennand is a New York Stem Cell Foundation - Robertson Investigator. The Brennand Laboratory is supported by a Brain and Behavior Young Investigator Grant, National Institute of Health (NIH) grant R01 MH101454 and the New York Stem Cell Foundation.

1. hiPSC Differentiation to Neural Progenitor Cells
<ol>
	<li>Grow and expand hiPSCs in human embryonic stem cell (HES) media (Table 1) co-cultured on a mouse embryonic fibroblast (mEF) feeder layer until large (but subconfluent) colonies are ready for neural differentiation via an embryoid body (EB) intermediate (Figure 2). Routine hiPSC culture conditions are well described elsewhere 16,17; briefly, grow hiPSCs in HES media on a mEF feeder layer until confluent, then enzymatically passage with collagenase (1mg/ml in DMEM) and expand approximately 1:3 every 7 days.</li>
	<li>To prepare Poly-L-Ornithine/laminin coated plates, coat plates with 10 &#956;g/ml Poly-L-Ornithine in sterile water for plastic surfaces (50 &#956;g/ml for glass surfaces) and incubate at RT for 3-24 hr. Wash at least once with sterile water and then coat with 5&#956;g/ml Laminin in sterile PBS at RT for 3-24 hr. 
	NOTE: If wrapped in plastic, plates can be stored for up to six months at -20 &#186;C.</li>
	<li>On Day 1, enzymatically lift subconfluent hiPSCs (generally grown on mEFs, or hiPSCs grown in defined media such as TeSR 18 on Matrigel) from the plate as large colonies using 1mg/ml Collagenase IV in DMEM/F12. 
	NOTE: Following 1-2 hr of incubation at 37 &#186;C, colonies will be floating in the dish.</li>
	<li>Gently wash hiPSC colonies (by settling, not centrifugation) 1-2x with DMEM/F12, resuspend in 2ml/well of N2/B27 media (Table 1) and transfer to non-adherent 6-well dishes (combine 3-wells into 1-well) 19. 
	NOTE: Overnight, colonies will form floating spherical clusters termed &#8220;embryoid bodies&#8221; (EBs). Expect substantial cellular death.</li>
	<li>On Day 2, tilt plates and allow EBs to settle. Carefully remove media and wash once with DMEM/F12 to remove debris. Feed with N2/B27 media supplemented with dual SMAD inhibitors (0.1 &#956;M LDN193189 and 10 &#956;M SB431542) 12.</li>
	<li>On Days 3-7, neuralization will occur in the context of dual SMAD inhibition; check that EBs are round but not cystic. Feed EBs every second day with N2/B27 media supplemented with 0.1 &#956;M LDN193189 and 10 &#956;M SB431542.</li>
	<li>On Days 7-14, following plating of EBs to Poly-L-Ornithine/Laminin coated plates, check using a brightfield microscope that neural rosettes begin to appear within a few days (characterized as round clusters of neuroepithelial cells with apico-basal polarity; Figure 1). Continue to feed adherent EBs every second day with N2/B27 media supplemented with 0.1 &#956;M LDN193189, 10 &#956;M SB431542 and 1 &#956;g/ml laminin.</li>
</ol>
2. Harvest of Neural Rosettes
NOTE: We recommend that neural rosettes be enzymatically harvested using Neural Rosette Selection Reagent 20 or similar selection reagent. Though neural rosettes can be manually picked into 6-well Poly-L-Ornithine/Laminin coated plate, this methodology takes extensive training to master, and, dependent on user skill, may require a second round of picking at day 20 to further enrich for NPCs and deplete non-neural cell types.
<ol>
	<li>For enzymatic selection of neural rosettes at day 14, aspirate media from adhered EBs and add 1 ml of neural rosette selection reagent per well for a 6-well plate. Incubate at 37 &#186;C for 1 hr.</li>
	<li>With a P1000 pipetman, gently remove enzyme from each well. Add 1 ml of DMEM/F12 per well to wash.</li>
	<li>With a P1000, collect the 1 ml of DMEM/F12 and quickly expel the DMEM/F12 back into the well, thus detaching the rosettes from the plate. Collect the rosettes into a falcon tube.</li>
	<li>Pipet another 1 ml of DMEM/F12 and quickly expel into the same well to detach remaining rosettes. (Do not triturate. Try not to break up rosette aggregates). Collect the neural rosettes into same falcon tube.</li>
	<li>Repeat step 2.4 as necessary. If rosettes do not detach readily, add more enzyme and repeat the process (steps 2.1-2.4)</li>
	<li>Spin cells at 300 x g for 3 min. Aspirate the wash, and re-suspend rosettes in 2 ml of NPC media (Table 1). Transfer cells (1:1) into a Poly-L-Ornithine/Laminin coated 6 well plate.</li>
	<li>From Days 15-21, the neural rosettes will expand; feed rosettes every second day with NPC media.</li>
	<li>Evaluate the quality of the neural rosettes and ensure that flat fibroblast-like cells are not expanding within the culture. 
	NOTE: If non-rosettes persist, the NPC culture can sometimes be salvaged by manual picking, selecting only the best of the picked rosettes into Accutase. Dissociate 15 min at 37 &#186;C. Pellet, wash and plate in NPC media onto 24-well Poly-L-Ornithine/Laminin coated plate.</li>
</ol>
3. Expansion of Neural Progenitor Cells
NOTE: hiPSC NPCs can be grown on either Matrigel- or Poly-L-Ornithine/Laminin coated plates. We typically use Matrigel-plates as they can be prepared more quickly and at lower cost.
<ol>
	<li>To prepare Matrigel coated plates, quickly thaw and resuspend a 1mg frozen aliquot of Matrigel in 24 ml cold DMEM/F12 and immediately distribute 2 ml to each well of two six-well plates. Incubate for at least 1 hr at 37 &#186;C. 
	NOTE: Matrigel needs only to be aspirated, not washed, immediately prior to use.</li>
	<li>Feed NPCs every second day and maintain at very high density or they will spontaneously differentiate to neurons. 
	NOTE: Though more established NPCs are typically split 1:4 every week, very low passage NPCs may be split 1:2 as infrequently as once every two weeks.</li>
	<li>To split, first aspirate media and add 1 ml warm Accutase (1X) per well of 6-well plate. Incubate at 37 &#186;C for 10-15 min.</li>
	<li>Gently transfer detached cells into a 15 ml tube containing DMEM/F12 with as little mechanical stress as possible. Do not titrate cells while in enzyme. Pellet cells by spinning at 1,000 x g for 5 min.</li>
	<li>Aspirate the wash, and re-suspend NPCs in ~1 ml NPC media per original well of 6-well. 
	NOTE: Expect that a confluent well of a 6-well dish has 5-10 million cells; this can be confirmed by counting a 10&#956;l aliquot of cells using a hematocytometer prior to re-plating.</li>
	<li>Following re-suspension, re-plate NPCs as NPCs, neurons or neurospheres. To maintain NPCs, plate approximately 1-2 million cells per well of a 6-well plate. The efficiency of neurosphere formation can vary between experiments and cell lines, but generally occurs best if between 200,000 and 1,000,000 cells are seeded per well of a non-adherent 6-well plate; if clumping of the neurospheres occurs, reduce the number of cells seeded. To differentiate to neurons, plate approximately 200,000 cells per well of a 6-well plate, replacing NPC media with Neuron media.</li>
	<li>Immunohistochemically validate every established NPC line. Once sufficient cellular material has been expanded, label NPCs with antibodies for NESTIN and SOX2. Validated NPC lines should also be differentiated into neurons for 4-6 weeks, and labeled with antibodies for &#946;III-TUBULIN and MAP2AB.
	<ol>
		<li>Fix cells in 4% paraformaldehyde in PBS at 4&#176;C for 10 min. Permeabilize NPCs at RT for 15 min in 1.0% Triton in PBS, and then block in 5% donkey serum with 0.1% Triton at RT for 30 min.</li>
		<li>Incubate with primary antibody in 5% donkey serum with 0.1% Triton, overnight at 4&#176;C. 
		NOTE: We recommend the following antibodies: goat anti-Sox2, 1:200; mouse anti-human Nestin, 1:200; rabbit anti-&#946;III-tubulin, 1:500; mouse anti-&#946;III-tubulin, 1:500; mouse anti-MAP2AB, 1:200. (Figure 2).</li>
		<li>Following a wash with PBS, incubate secondary antibodies cells with the appropriate conjugated secondary antibody to goat, mouse or rabbit at 1:300, in in 5% donkey serum with 0.1% Triton for 1-2 hr at RT. To visualize nuclei, stain cells with 0.5 &#956;g/ml DAPI (4&#39;,6-diamidino-2-phenylindole) and then mount with Vectashield.</li>
	</ol>
	</li>
</ol>
4. NPC Transduction
<ol>
	<li>Use spinfection (centrifugation of cell culture plates in the presence of virus at 1,000 x g for 1 hr) to increase the percentage of transfected cells 21. Aspirate media and replace with the relevant overexpression (or control) lentiviruses (or retroviruses), titered to the desired multiplicity of infection (MOI) (typically 1-10), diluted in NPC media. Use 1.5 ml volume per well of a 6-well plate (or an appropriately scaled volume for other types of tissue culture plates). Spin at 1,000 x g and 37 &#186;C for 1 hr in a plate centrifuge. 
	NOTE: Ideally, transduce NPCs with lentiviral or retroviral vectors within 1-2 days of splitting. Whether using commercial or laboratory-prepared viruses, it can be helpful to appropriately titer batches of virus in advance by serial dilution. (Figure 3).</li>
	<li>To reduce cellular death, replace media within 8 hr of spinfection. 
	NOTE: Viral integration and transgene expression should be detectable within 24 hr by reporter gene fluorescence. (If the virus lacks a fluorescent reporter, this is more difficult to assess; successful transfection can best be validated by immunoblot, immunohistochemistry or qPCR for overexpression products.) Full expression may take 3-7 days; repeated spinfections with additional virus may be required to increase the percentage of transfected cells. Recurrent spinfections can occur daily but need not be so frequent. Ideally, if using a fluorescent reporter, &#62;80% of cells should be labeled.</li>
</ol>
5. Neurosphere Migration Assay
NOTE: Neurospheres form spontaneously, following the enzymatic dissociation of NPCs (by a manner identical to that used in NPC expansion - steps 3.3-3.6), if cells are cultured in suspension in NPC media.
<ol>
	<li>Briefly, grow dissociated NPCs in NPC media for 48 hr in nonadherent plates in order to generate neurospheres. (Figure 4).</li>
	<li>For neurosphere migration, prepare fresh Matrigel plates using cold NPC media, rather than DMEM, in a 96-well plate, 1-2 hr prior to neurosphere picking. Do not aspirate Matrigel from the wells.</li>
	<li>As originally published for embryonic stem cell-derived neurospheres 22, manually pick NPC-derived neurospheres under a microscope, after washing once in NPC media to remove cellular debris. Transfer one neurosphere to each well of a Matrigel-coated 96-well plate.</li>
	<li>Add an additional 0.5 mg Matrigel, diluted in cold NPC media, to the neurospheres in each 96-well plate.</li>
	<li>Manually center each individual neurosphere in the middle of the well using a pipet tip. 
	NOTE: It is important to pick neurospheres of similar size, in order to reduce variability in the results.</li>
	<li>Allow migration to occur for 48 hr and then fix cells with 4% paraformaldehyde and stain with desired immunohistochemical markers.</li>
	<li>If radial migration is to be determined, photograph neurospheres in their entirely using a 4x microscope objective. Exclude any neurospheres contacting the edge of the well or a second neurosphere from the analysis. 
	NOTE: If migration occurs for longer than 48 hr, the resulting radial migration will likely be too large to image using a 4x objective.</li>
	<li>Measure average migration from each neurosphere using NIH ImageJ software (Figure 5). This can be done using either of the analysis methods described below:
	<ol>
		<li>Radial migration:
		<ol>
			<li>Manually trace the edge of the furthest migrating cells using the freehand selection tool then use the Measure (Ctrl+M) function to calculate the area of the resulting shape. Before measuring the area make sure that Area is checked in the Set Measurements window found under the Analyze tab. Use the value of the area measurement and the equation for the area of a circle (A=&#960;r2) to determine the radius (outer).</li>
			<li>Trace the edge of the original neurosphere, measure the area and calculate the radius (inner) in the same way. Calculate the total radial migration as the difference between the outer and inner radii.</li>
		</ol>
		</li>
		<li>Greatest cellular migration:
		<ol>
			<li>Measure the distance moved of the five furthest cells from the edge of the inner neurosphere using the straight-line selection tool followed by the Measure (Ctrl+M) function.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

We have described methods by which to differentiate hiPSCs into NPCs, a neural cell type in which a significant fraction of the gene signature of hiPSC-derived neurons is conserved and that may serve as a proxy for the developmental pathways potentially contributing to disease pathogenesis 8,11. Additionally, as we have detailed, NPCs are a robustly replicative and easily transduced neural population, which we believe may be suitable for molecular and biochemical studies of disease predisposition.
<p class...

Gene expression studies of neurons differentiated in vitro from human induced pluripotent stem cells (hiPSCs) by us 1 and others 2,3 indicate that hiPSC neurons resemble fetal rather than adult brain tissue. At present, hiPSC-based models may be more appropriate for the study of predisposition to, rather than late features of, neurological disease. We have previously reported that a significant fraction of the gene signature of schizophrenia hiPSC-derived neurons is conserved in schizophrenia hiPSC-derived&#160;neural progenitor cells (NPCs), indicating that NPCs may be a useful cell type for studying the molecular pathways contributing to schizophrenia 1. We and others have reported aberrant migration, increased oxidative stress and reactive oxygen species, sensitivity to sub-threshold environmental stresses and impaired mitochondrial function in schizophrenia hiPSC NPCs 1,4-6, as well as decreased neuronal connectivity and synaptic function in schizophrenia hiPSC neurons 5,7-10. If the molecular factors contributing to aberrant migration and/or oxidative stress in schizophrenia hiPSC NPCs also underlie the reduced neuronal connectivity in schizophrenia hiPSC-derived neurons, NPCs could be a robust and highly replicative neural population with which to study the mechanisms responsible for disease. Furthermore, because one can rapidly generate large numbers of cells and need not wait weeks or months for neuronal maturation, NPC-based assays are suitable for the study of larger patient cohorts and are more amenable to high throughput screening. We believe that hiPSC NPCs can serve as a proxy for the developmental pathways potentially contributing to disease pathogenesis, as has already been demonstrated in disorders as diverse as schizophrenia 1 and Huntington&#8217;s disease 11.
To differentiate NPCs from hiPSCs, initial neural induction is accomplished by dual-SMAD inhibition (0.1&#956;M LDN193189 and 10&#956;M SB431542) 12. By antagonizing BMP and TGF&#946; signaling with these small molecules, endoderm and mesoderm specification is blocked, accelerating neuronal differentiation and leading to the formation of visible neural rosettes within one week of plating. Neural patterning occurs early in this process, presumably during the period of neural rosette formation and immediately thereafter. In the absence of other cues, these primitive neural cells assume an anterior forebrain-like fate 13. Immediately following neural rosette formation, and ongoing throughout NPC expansion, forebrain NPCs are cultured with FGF2 8,14. They have dual lineage potential and can be differentiated to neural populations of 70-80% &#946;III-TUBULIN-positive neurons and 20-30% glial fibrillary acidic protein (GFAP)-positive astrocytes (Figure 1). The majority of forebrain hiPSC neurons are VGLUT1-positive, and so are presumably glutamatergic, although approximately 30% of neurons are GAD67-positive (GABAergic) 8.
NPCs are routinely passaged more than ten times in vitro, while maintaining consistent differentiation profiles, and without accumulating karyotype abnormalities. Groups have reported passaging NPCs more than 40 times 15, however, we find that beyond ten passages, NPCs show increased propensity for astrocyte differentiation. NPCs well-tolerate multiple freeze-thaws and can be transitioned to grow as neurospheres by simply culturing in non-adherent plates. NPCs are efficiently transduced by viral vectors, enabling rapid evaluation of the molecular and cellular consequences of genetic perturbation, and easily expandable to yield sufficient material for biochemical studies. Furthermore, because viral vectors permit robust over-expression and/or knockdown of disease-relevant genes, in either control or patient derived neural cells, one can use this platform to test the effect of genetic background on these manipulations. Though not suitable for synaptic or activity-based assays requiring mature neurons, NPCs may be a practical alternative for many straightforward molecular or biochemical analyses of patient-derived neural cells.

<table border="0" cellpadding="0" cellspacing="0" width="1174">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Name of Material/ Equipment</td>
			<td style="width:171px;">Company</td>
			<td style="width:216px;">Catalog Number</td>
			<td style="width:500px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">DMEM/F12</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#11330</td>
			<td>for HES media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">DMEM/F12</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#10565</td>
			<td style="width:500px;">for neural media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">KO-Serum Replacement</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#10828</td>
			<td style="width:500px;">Needs to be lot tested</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Glutamax</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#35050</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">NEAA</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">&#160;#11140</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">2&#8208;mercaptoethanol (55mM 1000x)</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">&#160;#21985-023</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">N2</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">&#160;#17502-048</td>
			<td style="width:500px;">Needs to be lot tested</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">B27-RA</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">&#160;#12587-010</td>
			<td style="width:500px;">Needs to be lot tested</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">FGF2</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#13256-029</td>
			<td style="width:500px;">Resuspend in PBS + 1% BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">LDN193189</td>
			<td style="width:171px;">Stemgent</td>
			<td style="width:216px;">#04-0074</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">SB431542</td>
			<td style="width:171px;">Stemgent</td>
			<td style="width:216px;">#04-0010</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">BDNF</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:216px;">#450-02</td>
			<td style="width:500px;">Resuspend in PBS + 0.1% BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">GDNF&#160;</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:216px;">&#160;#450-10</td>
			<td style="width:500px;">Resuspend in PBS + 0.1% BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Dibutyryl cyclic-AMP</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">&#160;#D0627</td>
			<td style="width:500px;">Resuspend in PBS + 0.1% BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">L-ascorbic acid</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">#A0278</td>
			<td style="width:500px;">Resuspend in H20</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">STEMdiff Neural Rosette Selection Reagent</td>
			<td style="width:171px;">Stemcell Technologies</td>
			<td style="width:216px;">&#160;#05832</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Accutase</td>
			<td style="width:171px;">Innovative Cell Technologies</td>
			<td style="width:216px;">AT-104</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Collagenase IV</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#17104019</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">CF1 mEFs</td>
			<td style="width:171px;">Millipore</td>
			<td style="width:216px;">#PMEF-CF</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Poly-L-Ornithine</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">P3655</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Laminin, Natural Mouse 1mg</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#23017-015</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">BD Matrigel</td>
			<td style="width:171px;">BD</td>
			<td style="width:216px;">#354230</td>
			<td style="width:500px;">Resuspend on ice in cold DMEM at 10mg/ml, use 1mg per two 6-well plate equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Tissue culture treated 6-well plates</td>
			<td style="width:171px;">Corning</td>
			<td style="width:216px;">3506</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Ultra low attachment 6-well plates</td>
			<td style="width:171px;">Corning</td>
			<td style="width:216px;">3471</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">goat anti-Sox2&#160;</td>
			<td style="width:171px;">Santa Cruz</td>
			<td style="width:216px;">sc&#173;17320</td>
			<td style="width:500px;">use at 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">mouse anti-human Nestin</td>
			<td style="width:171px;">Millipore</td>
			<td style="width:216px;">MAB5326</td>
			<td style="width:500px;">use at 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">rabbit anti-&#946;III-tubulin</td>
			<td style="width:171px;">Covance</td>
			<td style="width:216px;">PRB&#173;435P</td>
			<td style="width:500px;">use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">mouse anti-&#946;III-tubulin</td>
			<td style="width:171px;">Covance</td>
			<td style="width:216px;">MMS&#173;435P</td>
			<td style="width:500px;">use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">mouse anti-MAP2AB</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">M1406</td>
			<td style="width:500px;">use at 1:200</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Plate centrifuge</td>
			<td style="width:171px;">Beckman Coulter</td>
			<td style="width:216px;">Beckman Coulter Allegra X-14 (with SX4750 plate carrier)</td>
			<td style="width:500px;"></td>
		</tr>
	</tbody>
</table>

a guide to generating and using hipsc derived npcs for the study of neurological diseases

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long period of disease progression prior to the onset of symptoms. Because fibroblasts from patients and healthy controls can be efficiently reprogrammed into human induced pluripotent stem cells (hiPSCs), and subsequently differentiated into neural progenitor cells (NPCs) and neurons for the study of these diseases, it is now possible to recapitulate the developmental events that occurred prior to symptom onset in patients. We present a method by which to efficiently differentiate hiPSCs into NPCs, which in addition to being capable of further differentiation into functional neurons, can also be robustly passaged, freeze-thawed or transitioned to grow as neurospheres, enabling rapid genetic screening to identify the molecular factors that impact cellular phenotypes including replication, migration, oxidative stress and/or apoptosis. Patient derived hiPSC NPCs are a unique platform, ideally suited for the empirical testing of the cellular or molecular consequences of manipulating gene expression.

Neural rosettes can be identified morphologically, using a brightfield microscope, by their characteristic appearance as round clusters of neuroepithelial cells with apico-basal polarity (Figure 1). Though NPCs are typically cultured at very high cell density, immediately following passaging, slightly pyramidal-shaped soma and bipolar neurite structure is visible (Figure 1D). Validated NPCs express NESTIN and SOX2 in the majority of cells, though &#946;III-TUBULIN staining is also visibl...

Watch this Scientific Journal Video about A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases at JoVE.com

A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

This protocol describes how neural progenitor cells can be differentiated from human induced pluripotent stem cells, in order to yield a robust and replicative neural cell population, which may be used to identify the developmental pathways contributing to disease pathogenesis in many neurological disorders.

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long ...

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18.1K Views.  Icahn School of Medicine at Mount Sinai. This protocol describes how neural progenitor cells can be differentiated from human induced pluripotent stem cells, in order to yield a robust and replicative neural cell population, which may be used to identify the developmental pathways contributing to disease pathogenesis in many neurological disorders.

Video: A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to lessen the impact of debilitating conditions but remain elusive in many cases. Only by understanding the causes and pathology of diseases associated with aging, can scientists begin to identify potential therapeutic targets and develop strategies for intervention. The most common age-related conditions are neurodegenerative disorders such as Parkinson's disease and blindness. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Genome wide association studies have previously identified loci that are associated with increased susceptibility to this disease and identified two regions of interest: complement factor H (CFH) and the 10q26 locus, where the age-related maculopathy susceptibility 2 (ARMS2) and high-temperature requirement factor A1 (HtrA1) genes are located. CFH acts as a negative regulator of the alternative pathway (AP) of the complement system while HtrA1 is an extracellular serine protease. ARMS2 is located upstream of HtrA1 in the primate genome, although the gene is absent in mice. To study the effects of these genes, humanized knock-in mouse lines of Cfh and ARMS2, knockouts of Cfh, HtrA1, HtrA2, HtrA3 and HtrA4 as well as a conditional neural deletion of HtrA2 were generated. Of all the genetically engineered mice produced only mice lacking HtrA2, either systemically or in neural tissues, displayed clear phenotypes. In order to examine these mice thoroughly and systematically, an initial phenotyping schedule was established, consisting of a series of tests related to two main diseases of interest: AMD and Parkinson's. Genetically modified mice can be subjected to appropriate experiments to identify phenotypes that may be related to the associated diseases in humans. A phenotyping regimen with a mitochondrial focus is presented here alongside representative results from the tests of interest.

A reverse-genetics approach to understanding gene families associated with human disease is presented, using mouse as a model system, and the subsequent mouse phenotyping schedule is described. Because mice defective in a gene of interest, HtrA2, manifested Parkinsonian symptoms, the phenotyping regimen is focused on identifying neurological defects.

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to ...

Setting Up a Stroke Team Algorithm and Conducting Simulation-based Training in the Emergency Department - A Practical Guide

Time is of the essence when caring for an acute stroke patient. The ultimate goal is to restore blood flow to the ischemic brain. This can be&#160;achieved by either&#160;thrombolysis with recombinant tissue-plasminogen activator (rt-PA), the standard therapy for stroke patients who present within the first hours of symptom onset without contraindications, or by an endovascular approach, if a proximal brain vessel occlusion is detected. As the efficacy of both therapies declines over time, every minute saved along the way will improve the patient&#39;s outcome.
This critical situation requires thorough work and precise communication with the patient, the family and colleagues from different professions to acquire all relevant information and reach the right decision while carefully monitoring the patient. This is a high fidelity situation. In nonmedical high-fidelity environments such as aviation, Crew Resource Management (CRM) is used to enhance safety and team efficiency.
This guide shows how a Stroke Team algorithm, which is transferable to other hospital settings, was established and how regular simulation-based trainings were performed. It requires determination and endurance to maintain these time-consuming simulation trainings on a regular basis over the course of time. However, the resulting improvement of team spirit and excellent door-to-needle times will benefit both the patients and the work environment in any hospital.
A dedicated Stroke Team of 7 persons who are notified 24/7 by a collective call via speed dial and run a binding algorithm that takes approximately 20 min, was established. To train everybody involved in this algorithm, a simulation-based team training for all new Stroke Team members was conceived and conducted at monthly intervals. This led to a relevant and sustained reduction of the mean door-to-needle time to 25 min, and enhanced the feeling of stroke readiness especially in junior doctors and nurses.

Every min counts in acute stroke care. This guide shows how to establish a Stroke Team algorithm and enhance its performance with regular simulation training. The principles of Crew Resource Management (CRM) facilitate a straight workflow, reduce door-to-needle times and increase staff satisfaction.

Time is of the essence when caring for an acute stroke patient. The ultimate goal is to restore blood flow to the ischemic brain. This can ...

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This compound is currently under investigation as a PET tracer for demyelination. We recently described a novel chemical reaction to produce metafluorinated pyridines consisting of direct fluorination of a pyridine N-oxide and the utilization of this reaction for the radiochemical synthesis of [18F]3F4AP. In this article, we demonstrate how to produce this tracer using an automated synthesizer and an in-house made flow hydrogenation reactor. We also show the standard quality control procedures performed before releasing the radiotracer for preclinical animal imaging studies. This semi-automated procedure may serve as the basis for future production of [18F]3F4AP for clinical studies.

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

We demonstrate the semi-automated radiochemical synthesis of [18F]3F4AP and quality control procedures.

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This ...

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...