A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases
February 21st, 2015
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.
The overall goal of this procedure is to generate neuro progenitor cells or NPCs from human induced pluripotent stem cells or high PSCs as a platform to study the cellular and molecular mechanisms to contributing to neurological diseases. This is accomplished by first differentiating high PSCs using dual smad inhibitors into neural rosettes. The second step is to harvest these neural rosettes and allow them to grow into expandable N PC populations.
Next lentiviral transduction with a spin infection. Step to improve efficiency is used to manipulate gene expression. The final step is to grow the NPCs in suspension so that they spontaneously form neurospheres.
Ultimately, this platform can be used to assay a variety of cellular phenotypes. For example, measuring the effect of genetic manipulation on neuro migration. Though this method can provide insight into cellular mechanisms such as migration.
It can also be applied to study other biological processes such as replication, oxidative stress, apoptosis, and neurite outgrowth. Differentiation of human-induced pluripotent stem cells or high PSCs leads to the formation of visible neural rosettes, which are characterized as round clusters of neuro epithelial cells with aico basal polarity for the enzymatic selection of neural rosettes at day 14. Aspirate media from the adhered embryo bodies or ebs and add one milliliter of neural roset selection reagent per well of a six Well plate incubate at 37 degrees Celsius for one hour.
After one hour. Use a P 1000 pipette man to gently remove the enzyme from each well and add one milliliter of D-M-E-M-F 12 per well. To wash the rosettes, collect the one milliliter of D-M-E-M-F 12 and quickly expel it back into the well, thus detaching the rosettes from the plate.
Collect the rosettes into a falcon tube Pipette another one milliliter of D-M-E-M-F 12 and quickly expel it into the same well to detach the remaining rosettes. Do not tri and try not to break up the rosette aggregates. Collect the neural rosettes into the same falcon tube.
More of the enzyme can be added and this procedure repeated if the rosettes do not detach readily when all the neural rosettes have been collected. Spin the Falcon tube at 300 G for three minutes. Aspirate the wash and resus.
Suspend the rosettes in two milliliters of neural progenitor cell media. Transfer the cells into a poly L ornithine laminate coated six wall plate, and grow at 37 degrees Celsius for one week. In this laboratory, neural progenitor cells or NPCs are grown on matrigel plates, fed every second day and maintained at a very high density.
To split the NPCs, first aspirate the media and then add one milliliter of warm accutane per well of a six Well plate incubate at 37 degrees Celsius for 10 to 15 minutes. Gently transfer the detached cells into a 15 milliliter tube containing D-M-E-M-F 12 with as little mechanical stress as possible. Do not T tritrate the cells while in the enzyme pellet the cells by spinning at 1000 G for five minutes after centrifugation, aspirate the supernatant and resuspend the NPCs.
In about one milliliter of NPC media per original well of a six well plate, five to 10 million cells are expected per confluent well of a six well plate following resuspension and cell counting. Repl the NPCs plate approximately one to 2 million cells per well of a six well plate to maintain the NPCs. The efficiency of neurospheres formation varies between experiment and cell lines, but usually occurs best if between 200, 000 and a million cells are seated per well of a six wall nonadherent plate if clumping occurs, reduce the number of cells seated Ideally within one to two days of splitting.
The NP NPCs should be transduced with lentiviral or retroviral vectors to increase the percentage of transfected cells. Use spin infection. Aspirate the media from each well and replace with the relevant overexpression or control lentiviruses or retroviruses titered to the desired multiplicity of infection diluted.
In NPC media Use 1.5 milliliters per well of a six well plate spin at 1000 G and room temperature for one hour in a plate centrifuge. After the spin, place the plate back in the incubator to reduce cellular death. Replace the media within eight hours of spin infection.
Begin this procedure by washing the NPC derived NEUROSPHERES once in NPC media to remove cellular debris. Tilt the plate at a 45 degree angle and allow the neurospheres to settle and then remove as much media as possible without aspirating the neurospheres. Replace with fresh media.
Next, manually pick the NPC derived NEUROSPHERES under a microscope using a P 200 pipette transfer one neuros sphere to each well of a matrigel coated 96 well plate. It is important to pick neurospheres of similar sizes in order to reduce variability in the results. Add an additional 0.5 milligrams of matri gel diluted in cold NPC media to the neurospheres in each 96 well plate.
Using a pipette tip manually center each individual neuros sphere in the middle of the well Allow neuros sphere migration to occur for 48 hours. After fixing and staining the cells with the desired immunohistochemical markers, photograph the neurospheres in their entirety using a Forex microscope objective. To measure radial migration, use image J software.
Use the freehand selection tool to manually trace the edge of the furthest migrating cells. Make sure that area is checked in the set measurements window found under the analyze tab. And then use the measure function to calculate the area of the resulting shape.
Use the value of the area measurement and the equation for the area of a circle to determine the outer radius in the same manner, trace the edge of the original neuros sphere. Measure the area and calculate the inner radius. Calculate the total radial migration as the difference between the outer and inner radii.
These images show the stages of high PSC neural differentiation from high PSCs to embryo bodies, neural rosettes, NPCs, and neurons validated NPCs express, the NPC marker nesting and the neural stem cell transcription factor SOX two in most cells. Beta three tubulin staining is also visible in all NPC populations. NPCs also express the neural stem cell transcription factor PAC six and the four brainin progenitor marker.
TBR two, but not midbrain markers such as LMX one A and FOX A two NPCs can differentiate to 70 to 80%beta three tubulin positive neurons shown in green and 20 to 30%glial fibrillary acidic protein positive astrocytes shown in red, high quality high PSC. NPCs express neston and SOX two in most cells, whereas low quality NPCs have patches of SOX two negative and nest in negative cells only. The four week old neurons differentiated from high quality NPCs express map two AB and beta three turbulent.
Following successful transduction with a high titer lentiviral or retroviral vector. Greater than 80%of cells are labeled by the fluorescent reporter. Included in the vector neuros sphere generation yields a population of neurospheres of relatively homogenous size, which can remain healthy in culture for approximately one week.
With regular feeding neuros sphere migration occurs robustly in healthy neurospheres as illustrated by these brightfield images before and after 48 hours of migration in matrigel After its development. This technique paved the way for researchers in the field of schizophrenia to explore neuro migration in human induced PL ponent stem cell derived NPCs. Don't forget that working with a retrovirus can be potentially hazardous and all tissue culture work should be done in BL two plus conditions when virus is potentially present.
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Overview
This protocol describes the differentiation of neural progenitor cells (NPCs) from human induced pluripotent stem cells (hiPSCs) to study neurological diseases. The process involves generating NPCs that can be expanded and manipulated for research purposes.
Key Study Components
Area of Science
- Neuroscience
- Stem Cell Biology
- Neurological Disorders
Background
- Human induced pluripotent stem cells (hiPSCs) can differentiate into various cell types.
- Neural progenitor cells (NPCs) are crucial for studying brain development and disease.
- Understanding NPC differentiation pathways can provide insights into neurological disorders.
- This protocol aims to create a reliable source of NPCs for research.
Purpose of Study
- To generate NPCs from hiPSCs for studying neurological diseases.
- To explore cellular and molecular mechanisms involved in disease pathogenesis.
- To provide a platform for future research on neurodevelopmental disorders.
Methods Used
- Differentiation of hiPSCs using dual SMAD inhibitors into neural rosettes.
- Harvesting neural rosettes to grow expandable NPC populations.
- Lentiviral transduction with spin infection to manipulate gene expression.
- Growing NPCs in suspension to form neurospheres.
Main Results
- Successfully differentiated NPCs from hiPSCs.
- Established expandable NPC populations for research.
- Demonstrated the effectiveness of lentiviral transduction.
- NPCs formed neurospheres in suspension culture.
Conclusions
- This protocol provides a reliable method for generating NPCs from hiPSCs.
- NPCs can be used to investigate the mechanisms of neurological diseases.
- The approach can facilitate further research into therapeutic strategies.
Frequently Asked Questions
Executive Industry Relevance
This method enables biopharma R&D to model early neurodevelopmental events in human cells, providing a scalable platform to interrogate disease mechanisms prior to symptom onset. By generating expandable, genetically tractable NPCs from patient-derived hiPSCs, teams can de-risk target hypotheses through empirical testing of gene manipulation effects on cellular phenotypes such as migration, replication, oxidative stress, and apoptosis. This supports predictive confidence in target validation and informs go/no-go decisions in preclinical neuroscience programs.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by assessing molecular consequences of gene expression changes in a human-derived neural system.
- Operational Value: Provides a renewable NPC source for consistent phenotypic screening across genetic perturbations.
- Predictive Value: Supports mechanistic de-risking by linking genetic manipulation to quantifiable cellular outcomes relevant to neurological disease pathways.
Screening & Assay Development
- Scientific Value: Generates neurosphere-forming NPC populations suitable for high-content migration and stress-response assays.
- Operational Value: Enables standardized, scalable neurosphere production for reproducible compound or genetic screening campaigns.
- Assay Readiness: Produces expandable NPCs that can be cryopreserved, passaged, and transitioned to suspension culture for longitudinal phenotypic monitoring.
Translational & Preclinical Research
- Translational Continuity: Bridges discovery findings to preclinical validation by modeling human neurodevelopmental processes in a dish.
- Disease-Relevant System: Uses patient-derived NPCs to capture genetic background effects on cellular phenotypes, enhancing clinical relevance.
- Risk-Adjusted Advancement: Enables early assessment of target modulation effects on neuronal differentiation and glial fate, informing safety and efficacy profiling.
Pipeline & Workflow Integration
The method fits within the discovery continuum from target hypothesis generation through lead identification, providing a human cellular system to validate targets before committing to medicinal chemistry campaigns.
- Discovery Biology: Supports pathway clarification and target de-risking by enabling controlled manipulation of gene expression in NPCs and measurement of downstream phenotypic effects.
- Screening: Delivers assay-ready NPCs and neurospheres for quantifying impacts on migration, replication, oxidative stress, and apoptosis following genetic or pharmacological perturbation.
- Analytics: Generates imaging-based migration metrics (e.g., radial distance via ImageJ) and fluorescent reporter readouts to assess transduction efficiency and phenotypic changes.
- Translational Research: Maintains developmental and disease relevance by using patient-derived cells to model early pathogenic events in neurological disorders.
- Enterprise Reuse: Establishes a scalable NPC platform that can be reused across multiple targets, genetic backgrounds, and phenotypic assays within a neuroscience discovery portfolio.
Operational & Enterprise Impact
- Scientific Value: Increases target validation confidence by reducing reliance on post-mortem or non-human models through direct empirical testing in human neural progenitors.
- Operational Value: Ensures reproducibility through standardized NPC expansion, cryopreservation, and neurosphere formation protocols.
- Strategic Value: Improves capital efficiency by enabling early de-risking of targets, reducing late-stage failure due to unanticipated biological mechanisms in human neurons.
- Portfolio Impact: Facilitates data-driven prioritization of targets based on phenotypic rescue or exacerbation in a clinically relevant human cellular system.
Implementation Considerations
- Requires expertise in stem cell culture, neural differentiation, and lentiviral transduction techniques.
- Depends on access to poly-L-ornithine/laminin or Matrigel-coated surfaces, neural progenitor media, and centrifugation equipment for rosette harvesting and NPC splitting.
- Necessitates standardized protocols for neurosphere formation and migration assays to minimize variability across experiments and cell lines.
- Requires biosafety level 2-plus conditions when working with lentiviral or retroviral vectors to ensure safe handling.
- Success depends on careful handling to avoid rosette breakage during harvesting and maintaining optimal seeding densities for neurosphere formation.
