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

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.