Generation of Neuronal Cells from Blood-Derived Pluripotent Stem Cells

Ethic approvals were obtained when performing the experiments.

1. Isolation of human peripheral blood mononuclear cells (PBMNCs)

1. Ensure that all donors signed informed consent before blood withdrawing in compliance with institutional guidelines.
2. Take 30 mL of blood from healthy donors by trained medical personnel according to the standard protocol.
3. Isolate PBMNCs by density gradient media. Use 10 mL of media with 25 mL of 1:1 blood diluted with phosphate-buffered saline (PBS), and centrifuge at 300 x g for 30 min.
4. Isolate the interphase layer between the plasma and the density gradient media by pipetting. Wash the isolated cells with 5 mL of sterile PBS and centrifuge at 300 x g for 10 min. Repeat twice.
5. Count the number of cells by standard methods using a counting chamber.

2. Activation of human glycosylphosphatidylinositol (GPI)-anchored glycoprotein by antibody crosslinking on the surface of PBMNCs

1. Place the 6 x 10⁶ mononuclear cells (MNCs) in 15 mL tubes and perform antibody crosslinking by incubating the cells with human GPI-linked membrane protein-specific antibody (30 µg/mL) for 30 min in PBS with 1% bovine serum albumin (BSA) at 37 °C.
2. Replace incubation medium with Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum (FBS).
3. Grow cells in 15 mL polystyrene tubes, put the tubes in an incubator at 37 °C and 5% CO₂ for 8-10 days (without shaking). On day 5 (D5), add an additional 1-2 mL of Iscove's medium supplemented with 10% FBS to each 15 mL tube.

3. Sorting of newly generated dedifferentiated cells

1. Count cells with an automated cell counter (18 µL cell suspension + 2 µL fluorescence dye) or in a counting chamber.
2. Centrifuge cultured cell suspension (5-7 x 10⁶) at 300 x g for 10 min and aspirate the resulting supernatant with a sterile Pasteur pipette.
3. Re-suspend the cell pellet in 90 µL of pre-cooled PBS pH 7.2, 0.5% BSA and 2 mM ethylenediaminetetraacetic acid (EDTA).
4. Add CD45 positive nano-sized magnetic beads (80 µL) to the cell suspension and incubate on ice for 15 min.
5. Wash the cells by adding 2 mL of PBS buffer and centrifuge at 300 x g for 10 min.
6. Re-suspend the cells in 500 µL of PBS buffer.
7. Wash the column with 500 µL of pre-cooled PBS buffer and place it in the magnetic field.
8. Place the cell suspension on the column and wash it with 500 µL of PBS buffer (two times) and the centrifuge flow containing CD45 negative cells. Collect them in Iscove's medium supplemented with 1% BSA.
9. Count the cells in the counting chamber.

4. Preparing cell culture dishes for neuronal differentiation of newly generated stem cells

1. Coat the culture vessels with poly-L-ornithine and laminin for growing neuronal cells.
2. Place the glass coverslips in 4-well plates and coat it with 1:5 diluted poly-L-ornithine (0.1 mg/mL in double distilled water [ddH₂O]) in ddH₂O. Place the coverslips into a 37 °C incubator for 1 h. Then wash with ddH₂O.
3. Slowly thaw laminin (0.5-2.0 mg/mL) and add to the top of coverslips. Incubate it at 37 °C for 2 h.
4. Prepare neural induction medium N2 consisting of 49 mL of Dulbecco's modified Eagle medium/nutrient mixture F-12 (D-MEM/F12), 500 µL of N2 supplement, 400 µL of non-essential amino acids (NEAA), basic fibroblast growth factor (FGF) solution at 20 ng/mL final concentration (prepared from 100 µg/mL stock solution), and heparin at 2 ng/mL final concentration.
5. Remove excess laminin by pipetting and add neuronal medium N2 to culture dishes.

5. Culturing of neuronal dedifferentiated blood cells

1. Culture BD-derived CD45 negative cells on laminin/ornithine-coated glass coverslips for 2 days in an incubator at 37 °C and 5% CO₂ in N2 medium to initiate a neuronal differentiation of newly BD-generated cells.
2. Culture cells further in neuronal differentiation medium consisting of 48 mL of Neurobasal medium, 500 µL of L-glutamine, 1 mL of B27 Supplement, 500 µL of NEAA, 50 µL of recombinant human glial-derived neurotrophic factor (GDNF) at 5 µg/250 µL in PBS/0.1% BSA, and 50 µL of recombinant human brain derived neurotrophic factor (BDNF) at 5 µg/200 µL in PBS/0.1% BSA and 50 µL of ascorbic acid solution 2.9 g/50 mL in PBS. Place plates in an incubator at 37 °C and 5% CO₂.