GM-Free Generation of Blood-Derived Neuronal Cells

The significance of the protocol relies on membrane to nucleus signaling and does not directly affect the genome, like it is the case with induced pluripotent stem cells. The non-teratogenic nature of blood-derived pluripotent stem cells is the main advantage of the technique, making it suitable for safe clinical application in various field of regenerative medicine. Demonstrating the procedure is Dr.Anne-Kathrin Schott, project leader, and Oksana Greenacre, a lab assistant in my laboratory.

To isolate PBMNCs, add 25 milliliters of one-to-one blood diluted with PBS to 10 milliliters of density gradient media and centrifuge the mixture at 300 times G for 30 minutes. Isolate the interface layer between the plasma and the density gradient media by pipetting. Wash the isolated cells with five milliliters of sterile PBS and centrifuge at 300 times G for 10 minutes.

After repeating the procedure twice, count the number of cells using a counting chamber. Add 6 by 10 to the 6th mononuclear cells to a 15 milliliter tube, then perform antibody cross-linking by adding human GPI-linked membrane protein-specific antibody to the cells in PBS with 1%BSA and incubating for 30 minutes at 37 degrees Celsius. After the cross-linking, replace incubation medium with Iscove's Modified Dulbecco's Medium supplemented with 10%FBS.

Grow the cells in 15 milliliter polystyrene tubes by placing them in an incubator at 37 degrees Celsius and 5%carbon dioxide for 8 to 10 days without shaking. On the fifth day, add an additional one to two milliliters of Iscove's Medium supplemented with 10%FBS to each 15 milliliter tube. Count the cultured cells with a counting chamber.

Then centrifuge the cell suspension of approximately 5 by 10 to the 6th cells at 300 times G for 10 minutes and aspirate the resulting supernatant with a sterile Pasteur pipette. Resuspend the cell pellet in 90 microliters of pre-cooled PBS of pH 7.2 with 0.5%BSA and two millimolar EDTA. Then add 80 microliters CD-45 positive nano-sized magnetic beads to the cell suspension and incubate on ice for 15 minutes.

To wash the cells, add two milliliters of PBS buffer and centrifuge at 300 times G for 10 minutes. Resuspend the cells in 500 microliters of PBS buffer. Wash the column with 500 microliters of pre-cooled PBS buffer and place it in the magnetic field.

Place the cell suspension on the column and wash it twice with 500 microliters of PBS buffer. Collect the cells in Iscove's Medium supplemented with 1%BSA and count the cells in the counting chamber. Place glass coverslips in four-well plates and coat them with one to five diluted poly-L-ornithine and double distilled water.

After incubating the coverslips at 37 degrees Celsius for one hour, wash them with double distilled water. Slowly thaw 0.5 to two milligrams per milliliter of laminin and add it to the top of coverslips. Incubate them at 37 degrees Celsius for two hours, then remove excess laminin by pipetting and add neuronal medium N2 to the culture dishes.

Culture BD-derived CD-45 negative cells in N2 medium on laminin ornithine-coated glass coverslips for two days in an incubator with 5%carbon dioxide at 37 degrees Celsius to initiate a neuronal differentiation of newly BD generated cells. Next, culture cells in neuronal differentiation medium and place the plates in an incubator at 37 degrees Celsius and 5%carbon dioxide for 16 days. Prepare a fixative with 75 milliliters of sterile water and four grams of paraformaldehyde.

Add 10 normal sodium hydroxide as needed with stirring until the solution clears. Add sucrose mixture to the solution and titrate it to pH 7.4 with six normal hydrochloric acid. Bring the volume to 100 milliliters with sterile water.

After removing media from the cell culture, incubate it with prewarm fixative for 15 minutes, then discard the fixative and wash the cells three times for five minutes each. Immediately add a freshly made 0.3%Triton-X solution and permeabilize the cells for five minutes. Wash three times with PBS and add a blocking solution of PBS and 5%BSA.

Block the cells at room temperature on a rocker plate for one hour. Prepare an appropriate dilution of antibodies in 1%BSA PBS and incubate the cells with the antibodies on a rocker plate for 1.5 hours at room temperature. Wash the cells three times with PBS for five minutes per wash.

Incubate the cells with DAPI and mount the coverslips with mounting media for visualization in a microscope. The morphological aspect of BD dedifferentiated cells was studied on days one, five, and 10, showing a trend of gradual disappearance of nonactivated cells and a steadily growing new population of cells in activated culture. BD dedifferentiated cells were small and showed the characteristics of immature agranular cells with less organelles and large nuclei with condensed chromatin similar to ESCs.

Cells were studied for neuro re-differentiation after culturing in laminin-coated culture dishes for 30 days. As early as four days, the first neuronal-like cells with long branching structures could be detected. Most small spherical cells developed into branched cells with larger elongated shapes, implying an active process toward differentiation to neuronal lineages.

The cell body and processes of the cells showed a higher complexity than undifferentiated cells, presenting a high density of rough endoplasmic reticulum and bundles of actin filaments. Cells growing in differentiation media frequently established cell-to-cell contacts involving the cellular body, while others involved cellular processes in neurite-like fashion. Immunochemistry analysis performed using specific antibodies confirmed that BD dedifferentiated cells are capable of re-differentiation towards various neuronal lineages, including GFAP astrocytes.

This technique make it possible to create autologous non-teratogenic cells capable of re-differentiation into cells of all three germ layer, opening new opportunity in regenerative medicine research.