Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

This protocol describes a method to reprogram blood and mononuclear cells to neural stem cells that can be used to treat neurodegenerative diseases, such as Parkinson's disease. It offers an autologous cell source to treat neurodegenerative diseases. Also, the time frame required for conversion to induced neural stem cells and differentiation to desired cell types is shorter than for induced pluripotent stem cells.

Induced neural stem cells possess a good proliferative capacity and can be scaled up before application. Since the iNSCs can be specified into different region-specific neural cells, such as spinal cord neurons or motor neurons, this method may be extended for the treatment of other neurological diseases. The whole procedure takes a long time, with all steps related and critical, so visual demonstration gives an idea about how the cells look like and provides some tips to ensure a successful generation of the desired cells.

To begin, transfer previously isolated peripheral venous blood into a 50-milliliter conical tube, and dilute it with an equal volume of sterile Dulbecco's PBS. In another 50-milliliter conical tube, prepare 15 milliliters of sterilized density gradient medium. Tilt the tube at a 45-degree angle, and slowly and carefully lay 30 milliliters of diluted peripheral blood onto the density gradient medium.

Centrifuge the tube at 800 times g for 15 minutes at room temperature with the centrifuge brake set at the off position. Aspirate the yellow, upper plasma layer, and discard it. Use a 10-milliliter pipette to transfer the white, cloudy thin film layer containing mononuclear cells to a new 50-milliliter conical tube.

Add 30 milliliters of D-PBS to the tube with MNCs, and centrifuge at 600 times g for 10 minutes at four degrees Celsius. After discarding the supernatant, add 45 milliliters of D-PBS and resuspend the cells. After centrifuging the tube at 400 times g for 10 minutes at four degrees Celsius, discard the supernatant.

Resuspend the cells with five milliliters of D-PBS, count the live cells with the trypan blue exclusion method, set aside the MNCs needed for expansion, and freeze the remaining cells for future use. On day minus 14, seed MNCs at a density of two to three million cells per milliliter per well of six-well plate with 1.5 milliliters of 37-degree Celsius, pre-warmed MNC medium. Incubate at 37 degrees Celsius, 5%carbon dioxide for two days.

On day minus 11, use a sterilized pipette to collect the cells with the medium and transfer to a new 15-milliliter conical tube. Centrifuge at 250 times g for five minutes at room temperature, discard the supernatant, and resuspend the cells in one milliliter of pre-warmed MNC medium. After counting the viable cells with trypan blue, seed the MNCs at a density of one million cells per milliliter in pre-warmed MNC medium in a six-well plate.

Incubate at 37 degrees Celsius, 5%carbon dioxide for three days, and repeat cell collecting, centrifuging, and plating in MNC medium on days minus eight and minus four. Sendai virus in this procedure is hazardous, and all procedures involving Sendai virus must be performed in a safety cabinet. And all tips and tubes exposed to Sendai virus should be treated with ethanol or bleach before disposal.

On day zero, collect the cells in MNC medium and transfer to a 15-milliliter conical tube. After centrifuging the cells at 200 times g for five minutes, aspirate the supernatant and resuspend the cells with one milliliter of pre-warmed MNC medium. Count the viable cells with trypan blue, and then resuspend them with pre-warmed MNC medium to a concentration of 200, 000 cells per well in 24-well plates.

Thaw the tubes removed from minus 80-degree Celsius storage containing Sendai virus in a 37-degree Celsius water bath for five to 10 seconds, and then leave them to thaw at room temperature. Once thawed, place them immediately on ice. Add the thawed Sendai virus to the wells of the 24-well plate with MNCs at a multiplicity of infection of 10.

To facilitate the attachment of cells, centrifuge the plates at 1, 000 times g for 30 minutes and after that place the plates in the incubator at 37 degrees Celsius, 5%carbon dioxide. On day one, transfer the cells with the medium to a 15-milliliter centrifuge tube. To further detach the remaining cells, rinse the wells with one milliliter of MNC medium per well and add the cells with medium to the tube.

After centrifuging the cell suspension at 200 times g for five minutes, aspirate the supernatant, resuspend the cells with 500 microliters of fresh pre-warmed MNC medium, and add to a well of a 24-well plate. On day two, use one milliliter per well of previously diluted 50 micrograms per microliter poly-D-lysine in D-PBS to coat six-well plates for at least two hours at room temperature. Aspirate poly-D-lysine from the six-well plates, and dry on the vertical clean bench for about 10 minutes.

Then add one milliliter per well of a previously diluted five micrograms per milliliter laminin to the six-well plates, and incubate for four to six hours at 37 degrees Celsius to coat. Wash the plates with D-PBS before use. On day three, plate all Sendai virus-transduced MNCs in induced neural stem cell medium on the poly-D-lysine/laminin-coated six-well plates.

On days five and seven, gently add one milliliter of 37-degree Celsius, pre-warmed iNSC medium to each well of the six-well plates. From day nine to day 28, replace the medium with fresh pre-warmed iNSC medium daily. Monitor the emergence of iNSC colonies, and in about two to three weeks pick colonies with appropriate morphology using burned glass pipettes and transfer each colony to a separate well of a six-well plate for expansion.

To establish unilateral 6-hyroxydopamine-lesioned mouse models, shave the head of an anesthetized, adult, male, SCID-beige mouse, and apply erythromycin eye ointment. Place the mouse on the stereotaxic apparatus, and fix the mouse with incisor bars. Insert the ear cups correctly to securely and appropriately position the mouse head.

Sterilize the head with povidone iodine and isopropyl alcohol, and then use a scalpel blade to make an approximately 1.5-centimeter sagittal incision on the head skin, exposing the skull. Adjust the incisor bar and ear bars to reduce the height difference between bregma and lambda to less than 0.1 millimeters. Slowly move and lower the tip of the microsyringe needle towards bregma, and treat bregma as the zero point.

Move the tip to a position with coordinates of anterior 0.5 millimeters and lateral 2.1 millimeters relative to bregma. Retract the tip, mark the point with a marker pen, and, with an electronic drill, burr a little hole into the skull. Extract two microliters of five micrograms per milliliter 6-hyroxydopamine solution into the microsyringe.

Return the needle to the point marked, and insert the needle to dorsal/ventral 3.2 millimeters. Inject the 6-hyroxydopamine solution from the syringe at a rate of one microliter per minute, leave the injection needle in place for another five minutes, and then retract it slowly. After closing the incision with sutures, apply erythromycin eye ointment on the eyes again.

Remove the mouse from the stereotaxic apparatus, put it in the recovery cage, and allow access to food and water until it regains consciousness. After PBMNCs were infected with SeV, their morphology was changing and cells underwent a drastic death until day five. INSC colonies emerged on day 12.

After picking and transferring iNSC clones for expansion for a number of passages, they showed a good morphology and could self-renew stably in iNSC medium, either in a monolayer form or as spheres. After 24 days of differentiation, iNSCs were identified as dopaminergic neurons as a majority of them expressed tyrosine hydroxylase, forkhead box A2, and neuron-specific class III beta-tubulin markers. After establishment of unilateral 6-hyroxydopamine-lesioned Parkinson's disease mouse models, behavioral assessment was conducted to estimate Parkinson's disease symptoms.

The mice that had received cell transplantation showed significant improvement in motor function. The extent of 6-hyroxydopamine-induced lesioning was verified by post-mortem immunofluorescent staining for TH at the striatum, medial forebrain bundle, and substantia nigra pars compacta. The TH-positive signals were greatly recovered in the striatum where cells were implanted and also mildly recovered at SN.Three months after transplantation, among surviving cells, about 13.84%were TH-positive dopaminergic neurons.

About 91.72%of the TH-positive cells expressed orphan nuclear receptor, about 86.76%expressed FOXA2, and about 98.77%were co-labeled with G-protein-coupled inward rectifier potassium. When laying 30 mils of diluted peripheral blood onto the density gradient medium, please do it slowly and carefully, not to disturb the gradient interface. Care should be taken to avoid repeated thawing and freezing of Sendai virus to ensure a high virus activity.

When reprogramming peripheral blood mononuclear cells to induced neural stem cells, please closely observe the morphology of cells every day. Also, 6-hydroxydopamine is light-and temperature-sensitive. Be careful to protect the solution from light, and keep it on ice before use.

Following this procedure, one can continue to study the disease mechanisms by using familial PD patient-derived dopaminergic neurons in culture. One can also continue to investigate how the transplanted cells influence the damaged neuronal circuits.