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><tbody><tr><td>DMEM/F12</td><td>Life Technologies</td><td>#11330</td><td>for HES media</td></tr><tr><td>DMEM/F12</td><td>Life Technologies</td><td>#10565</td><td>for neural media</td></tr><tr><td>KO-Serum Replacement</td><td>Life Technologies</td><td>#10828</td><td>Needs to be lot tested</td></tr><tr><td>Glutamax</td><td>Life Technologies</td><td>#35050</td><td></td></tr><tr><td>NEAA</td><td>Life Technologies</td><td>&amp;nbsp;#11140</td><td></td></tr><tr><td>N2</td><td>Life Technologies</td><td>&amp;nbsp;#17502-048</td><td>Needs to be lot tested</td></tr><tr><td>B27-RA</td><td>Life Technologies</td><td>&amp;nbsp;#12587-010</td><td>Needs to be lot tested</td></tr><tr><td>FGF2</td><td>Life Technologies</td><td>#13256-029</td><td>Resuspend in PBS + 1% BSA</td></tr><tr><td>LDN193189</td><td>Stemgent</td><td>#04-0074</td><td></td></tr><tr><td>SB431542</td><td>Stemgent</td><td>#04-0010</td><td></td></tr><tr><td>BDNF</td><td>Peprotech</td><td>#450-02</td><td>Resuspend in PBS + 0.1% BSA</td></tr><tr><td>GDNF&amp;nbsp;</td><td>Peprotech</td><td>&amp;nbsp;#450-10</td><td>Resuspend in PBS + 0.1% BSA</td></tr><tr><td>Dibutyryl cyclic-AMP</td><td>Sigma</td><td>&amp;nbsp;#D0627</td><td>Resuspend in PBS + 0.1% BSA</td></tr><tr><td>L-ascorbic acid</td><td>Sigma</td><td>#A0278</td><td>Resuspend in H&lt;sub&gt;2&lt;/sub&gt;0</td></tr><tr><td>STEMdiff Neural Rosette Selection Reagent</td><td>Stemcell Technologies</td><td>&amp;nbsp;#05832</td><td></td></tr><tr><td>Accutase</td><td>Innovative Cell Technologies</td><td>AT-104</td><td></td></tr><tr><td>Collagenase IV</td><td>Life Technologies</td><td>#17104019</td><td></td></tr><tr><td>CF1 mEFs</td><td>Millipore</td><td>#PMEF-CF</td><td></td></tr><tr><td>Poly-L-Ornithine</td><td>Sigma</td><td>P3655</td><td></td></tr><tr><td>Laminin, Natural Mouse 1 mg</td><td>Life Technologies</td><td>#23017-015</td><td></td></tr><tr><td>BD Matrigel</td><td>BD</td><td>#354230</td><td>Resuspend on ice in cold DMEM at 10 mg/ml, use 1 mg per two 6-well plate equivalent</td></tr><tr><td>Tissue culture treated 6-well plates</td><td>Corning</td><td>3506</td><td></td></tr><tr><td>Ultra low attachment 6-well plates</td><td>Corning</td><td>3471</td><td></td></tr><tr><td>goat anti-Sox2&amp;nbsp;</td><td>Santa Cruz</td><td>sc&amp;shy;17320</td><td>use at 1:200</td></tr><tr><td>mouse anti-human Nestin</td><td>Millipore</td><td>MAB5326</td><td>use at 1:200</td></tr><tr><td>rabbit anti-&amp;beta;III-tubulin</td><td>Covance</td><td>PRB&amp;shy;435P</td><td>use at 1:500</td></tr><tr><td>mouse anti-&amp;beta;III-tubulin</td><td>Covance</td><td>MMS&amp;shy;435P</td><td>use at 1:500</td></tr><tr><td>mouse anti-MAP2AB</td><td>Sigma</td><td>M1406</td><td>use at 1:200</td></tr><tr><td>Plate centrifuge</td><td>Beckman Coulter</td><td>Beckman Coulter Allegra X-14 (with SX4750 plate carrier)</td><td></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...

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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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Medicine

18.2K 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

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

Developmental Biology

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and neurite outgrowth; and later stages characterized by axon/dendrite outgrowth and synapse formation. In neurodevelopmental disorders, often one or more of these processes are disrupted, leading to abnormalities in brain formation and function. With the advent of human induced pluripotent stem cell (hiPSC) technology, researchers now have an abundant supply of human cells that can be differentiated into virtually any cell type, including neurons. These cells can be used to study both normal brain development and disease pathogenesis. A number of protocols using hiPSCs to model neuropsychiatric disease use terminally differentiated neurons or use 3D culture systems termed organoids. While these methods have proven invaluable in studying human disease pathogenesis, there are some drawbacks. Differentiation of hiPSCs into neurons and generation of organoids are lengthy and costly processes that can impact the number of experiments and variables that can be assessed. In addition, while post-mitotic neurons and organoids allow the study of disease-related processes, including dendrite outgrowth and synaptogenesis, they preclude the study of earlier processes like proliferation and migration. In neurodevelopmental disorders, such as autism, abundant genetic and post-mortem evidence indicates defects in early developmental processes. Neural precursor cells (NPCs), a highly proliferative cell population, may be a suitable model in which to ask questions about ontogenetic processes and disease initiation. We now extend methodologies learned from studying development in mouse and rat cortical cultures to human NPCs. The use of NPCs allows us to investigate disease-related phenotypes and define how different variables (e.g., growth factors, drugs) impact developmental processes including proliferation, migration, and differentiation in only a few days. Ultimately, this toolset can be used in a reproducible and high-throughput manner to identify disease-specific mechanisms and phenotypes in neurodevelopmental disorders.

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells NPCs

Neurodevelopmental processes such as proliferation, migration, and neurite outgrowth are often perturbed in neuropsychiatric diseases. Thus, we present protocols to rapidly and reproducibly assess these neurodevelopmental processes in human iPSC-derived NPCs. These protocols also allow the assessment of the effects of relevant growth factors and therapeutics on NPC development.

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and ...

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo.
Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

Immunology and Infection

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.

Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

Neuroscience

A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides reliable analysis of neuronal morphology together with quantitative measurements of modifications on neurite length and synaptic protein localization and abundance upon treatment with small molecule compounds. In addition to the application of the presented method in neurite outgrowth studies, neurotoxicity assessment can be performed to assess, distinguish and rank commercial chemical compounds based on their potential developmental neurotoxicity effect.
Even though cell lines are nowadays widely used in compound screening assays in neuroscience, they often differ genetically and phenotypically from their tissue origin. Primary cells, on the other hand, maintain important markers and functions observed in vivo. Therefore, due to the translation potential and physiological relevance that these cells could offer neurite outgrowth assay and neurotoxicity assessment can considerably benefit from using human neural progenitor cells (hNPCs) as the primary human cell model.
The presented method herein can be utilized to screen for the ability of compounds to induce neurite outgrowth and neurotoxicity by taking advantage of the human neural progenitor cell-derived neurons, a cell model closely representing human biology.&#34;

The presented protocol describes a method for a neurite outgrowth assay and neurotoxicity assessment of small molecule compounds.

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides ...

Differentiation of Porcine Induced Pluripotent Stem Cells (piPSCs) into Neural Progenitor Cells (NPCs)

iPSC-derived neurons are attractive in vitro models to study neurogenesis and early phenotypic changes in mental illness, mainly when most animal models used in pre-clinical research, such as rodents, are not able to meet the criteria to translate the findings to the clinic. Non-human primates, canines, and porcine are considered more adequate models for biomedical research and drug development purposes, mainly due to their physiological, genetic, and anatomical similarities to humans. The swine model has gained particular interest in translational neuroscience, enabling safety and allotransplantation testing. Herein the generation of porcine iPSCs is described along with its further differentiation into neural progenitor cells (NPCs). The generated cells expressed NPC markers Nestin and GFAP, confirmed by RT-qPCR, and were positive for Nestin, b-Tubulin III, and Vimentin by immunofluorescence. These results show the evidence for the generation of NPC-like cells after in vitro induction with chemical inhibitors from a large animal model, an interesting and adequate model for regenerative and translational medicine research.

Differentiation of Porcine Induced Pluripotent Stem Cells piPSCs into Neural Progenitor Cells NPCs

This protocol describes a method for chemical differentiation and culture of neural progenitor cells derived from porcine induced pluripotent stem cells (piPSCs).

iPSC-derived neurons are attractive in vitro models to study neurogenesis and early phenotypic changes in mental illness, mainly when most animal models ...

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 ...

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 ...

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 ...

Production of Human Neurogenin 2-Inducible Neurons in a Three-Dimensional Suspension Bioreactor

The derivation of neuronal lineage cells from human induced pluripotent stem cells (hiPSCs) marked a milestone in brain research. Since their first advent, protocols have been continuously optimized and are now widely used in research and drug development. However, the very long duration of these conventional differentiation and maturation protocols and the increasing demand for high-quality hiPSCs and their neural derivatives raise the need for the adoption, optimization, and standardization of these protocols to large-scale production. This work presents a fast and efficient protocol for the differentiation of genetically modified, doxycycline-inducible neurogenin 2 (iNGN2)-expressing hiPSCs into neurons using a benchtop three-dimensional (3D) suspension bioreactor.
In brief, single-cell suspensions of iNGN2-hiPSCs were allowed to form aggregates within 24 h, and neuronal lineage commitment was induced by the addition of doxycycline. Aggregates were dissociated after 2 days of induction and cells were either cryopreserved or replated for terminal maturation. The generated iNGN2 neurons expressed classical neuronal markers early on and formed complex neuritic networks within 1 week after replating, indicating an increasing maturity of neuronal cultures. In summary, a detailed step-by-step protocol for the fast generation of hiPSC-derived neurons in a 3D environment is provided that holds great potential as a starting point for disease modeling, phenotypic high-throughput drug screenings, and large-scale toxicity testing.

This article describes a protocol for the generation of human induced pluripotent stem cell-derived neurons in a benchtop 3D suspension bioreactor.

The derivation of neuronal lineage cells from human induced pluripotent stem cells (hiPSCs) marked a milestone in brain research. Since their first ...

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

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