末梢血造血前駆細胞からヒト神経幹細胞の直接誘導

The overall goal of this procedure is to directly induce neural stem cells from blood cells. This is accomplished by first enriching CD 34 positive hematopoietic progenitor cells from blood. The second step is to transfect CD 34 cells with sendi virus containing transcriptional factors.

Next, the neural stem cells are derived using selective medium. The final step is to differentiate the induced neural stem cells into neurons, astroglia, and oligodendrocytes. Ultimately, immunofluorescence microscopy is used to characterize the resulting cells.

The main advantage of this technique over existing method, such as duration of neurons directly from fibroblast, also IPS generation, is that this method bypasses the tedious process of IPS generation and calculation, but still provide a large amount of neuronal cells which can relate, differentiate to neurons. I first had the idea for this method when I noticed that attached transfected CT 34 cells actually shows very similar to primary cultured neuro progenic cells demonstrating the procedure will be Marines, a technician from my life. After isolating CD 34 cells from peripheral blood mononuclear cells according to instructions in the text protocol, begin the process of culturing the CD 34 cells by Resus, suspending the CD 34 cells in CD 34 media then seed the cells into a 24 well plate at three times 10 to the fifth cells per well in one milliliter of medium.

After incubating for 24 hours in a 37 degree Celsius tissue culture incubator with 5%carbon dioxide, collect the floating cells and centrifuge the cell suspension at 110 times G at room temperature. Then discard the supernatant and resuspend the cells in fresh CD 34 maintenance medium. See a new 24 well plate at one times 10 to the fifth cells per well in one milliliter of medium, incubate at 37 degrees Celsius in a 5%carbon dioxide incubator for five additional days, replacing half of the medium every other day.

By carefully aspirating the top half of supernatant while the CD 34 cells are on the bottom of the wells as shown here on the fifth day. After plating, collect CD 34 cells and centrifuge the cells at 170 times G for 10 minutes at room temperature, discard the supernatant, resuspend the cells as before and seed a fresh 24 well plate at one times 10 to the fifth cells per well in one milliliter of media. Next thaw a cyto tune IPS Sendi reprogramming kit.

By first immersing the bottoms of the tubes in a 37 degrees Celsius water bath for 10 seconds and then completely thaw at room temperature. Briefly centrifuge the tubes and put them on ice. Prepare the Sendai virus mixture by mixing the recommended volume of each virus listed on the certificate of analysis for the corresponding lot.

A multiplicity of infection of 15 is recommended. Add the Sendai virus mix to each well of CD 34 cells. Then mix the wells by gently shaking the plate.

Incubate the cells at 37 degrees Celsius in a 5%carbon dioxide incubator after 24 hours. Observe the transduction efficiency. Notice that cell aggregates and spheres such as those shown here are indicators of successful transduction.

Then incubate the cells in the tissue culture incubator for five to seven days until attached cells appear and reach 30 to 50%confluence as shown here every other day. Change half of the medium by carefully transferring the top half of the medium to another well. If cell spheres are transferred to the new, well add an equal amount of fresh medium.

Check the co fluency of the cells using the inverted microscope. When the cells are 30 to 50%confluent, aspirate the supernatant and reserve and then add one milliliter of neural progenitor medium to each well to make a backup plate using the spheres in the supernatant centrifuge, the reserve supernatant at 170 times G for 10 minutes. Then aspirate the supernatant and resuspend the pellet in neural progenitor medium plate in a matrigel coated 24 well plate.

As before, change the medium in each plate of cells every other day until the cells reach 60 to 80%confluence. Usually after one week. Once the cells reach the required confluence, aspirate the supernatant and add one milliliter of neural stem cell medium to each.

Well then dissociate the cells by using a cell scraper, followed by very gentle pipetting. Next, transfer the cells from one well of the 24 well plate into one well of a six. Well plate.

Add another milliliter of medium to the well and after repeating the procedure for all wells, place the six well plate into the 37 degrees Celsius 5%carbon dioxide incubator. Change the full volume of medium every other day until the cells reach 60%confluence after which time dissociate and reflate the cells at a one to three ratio as just shown to freeze a portion of undifferentiated neural stem cells. First, prepare the stem cell freezing medium by adding 20%dimethyl sulf oxide or DMSO to the neural stem cell medium and place on ice.

Then dissociate the cells in the six well plate. Using a cell scraper and gentle pipetting. Reserve an aliquot of the suspension for a cell count and then centrifuge the cells at 170 times G for 10 minutes at room temperature while the cells are spinning, count the cells in the aliquot of suspension using a hemo, cytometer or automated cell counter and calculate the volume of medium required to give a cell concentration of one times 10 to the six cells per milliliter.

After the spin has finished, discard the supernatant and resuspend the pellet in neural stem cell medium at one times 10 to the six cells per milliliter. Then add an equal volume of freezing medium pipette five times 10 to the fifth cells in a volume of one milliliter into a cryo vial and freeze the cells in a freezing container according to the manufacturer’s instructions. When the neural stem cells in the six well plate reach 80%confluence, detach them using a rubber policeman and then dissociate by gentle pipetting.

Pool the cell suspension into a sterile tube. Count the cells using a hemo, cytometer or automated cell counter and adjust the concentration of the cell suspension to one times 10 to the fifth cells per milliliter with neural stem cell medium. Then pipette one milliliter of the cell suspension onto a poly D lysine and laminate coated cover slip in one well of a 24 well plate after repeating the procedure for the desired number of wells.

Incubate as before for 24 hours. After the incubation time has elapsed, aspirate the media and replace with neural differentiation. Medium incubate for two weeks at 37 degrees Celsius and 5%carbon dioxide changing the medium twice a week after two weeks.

The neuronal stem cells should be fully differentiated as shown here and ready for downstream applications such as immunofluorescence staining. The first two images show that neural stem cells express nest in and SOX two. While the third image demonstrates that OCT four is not expressed, DPI is used as a nuclear marker and the scale bar represents 200 microns.

After following the differentiation protocol outlined in the video, the differentiated neurons express the neuronal marker beta three tubulin. The human neural stem cells can also be differentiated into glial cells. When differentiated into astrocytes, the cells express the astrocytic marker GFAP.

Finally, in this image, the cells have been differentiated into oligodendrocytes and express the oligodendrocyte marker. O four. Once mastered the generation of induced human neural stem cells from peripheral blood can be done in approximately three weeks.

If performed properly, The neuro stem cells could be further differentiated neurons and unclear the method by is the lengthy and complicated IPS generation processes and could be a easy option to generate patient specific neuronal cultures after its development. This technique path way for researchers in the field of neuroscience to explore individual cell culture modeling for various neurological disorders, which might better represent the genetic differences in specific disorders, especially from individual patient samples.