The overall goal of this procedure is to induce highly efficient dopaminergic neuron differentiation. In vitro. This is accomplished by first preparing a single cell adhesion culture of human P pluripotent stem cells.
In the second step, the cell culture media is switched to neuron induction media to induce floorplate progenitors. Next, the dopaminergic neurons are cultured in maturation media. And then finally, the single cell dopaminergic neuron progenitors are cultured on poly L ornithine laminate fibronectin plates, and maturation media for 30 days.
Ultimately, immunofluorescence microscopy can be used to assess the expression of important dopaminergic neuron markers. This technique has a greater advantage over the existing neuro sphere on neuro acid protocol. It can generate more homogeneous double neural population with higher efficiency.
Four days after passage aspirate the medium from the cell cultures. Detach the cells with one milliliter of Accutane per well and incubate them at 37 degrees Celsius for five minutes. During the incubation, add one milliliter of gelatin to each well of a six well plate, and then place the plate along with freshly prepared matri gel coated plates into the incubator.
When the cells have become semi floating, pipette them up and down several times to make a single cell suspension. Then pull the cells into a 15 milliliter tube and centrifuge them for three minutes. At 258 Gs and room temperature, re suspend the pellet in the appropriate amount of human pluripotent stem cell culture medium supplemented with serum and add thi vivin to a final concentration of two millimolars.
Next, incubate the cells in the gelatin coated plates at a one-to-one ratio at 37 degrees Celsius for 30 minutes. To remove the meth feeders, then transfer the cells to a new 15 milliliter tube. Dilute them in human pluripotent stem cell medium, supplemented with serum, and count them after spinning down the cells again, re suspend the cells in MT.SR one medium with thias Ivan to a 180, 000 cells per milliliter concentration.
Then aspirate the matrigel from the coated plates and dispense two milliliters of cells per well. 48 hours. After replating the cells into the matrigel plates.
Aspirate the medium from each well and wash the cells once with PBS. Then add two milliliters of KSR differentiation, medium supplemented with SB 4 31, 50 42 an L DN 1 9 3 1 8 9 to the cells changing the medium every day from D zero to D 20 as outlined in the chart on day 20, aspirate the differentiation medium and detach the cells with one milliliter of 37 degrees Celsius acutus per well at 37 degrees Celsius for five minutes. During the incubation, warm poly L orine laminin fibronectin plates in the incubator.
Then after Resus, suspending the detached cells into a single cell suspension, dilute the cells in neuro basal media. After counting, spin down the cells and resuspend the pellet in B 27, differentiation medium at a 1 million cells per milliliter concentration. Then aspirate the fibronectin from the polyol orine laminin fibronectin plates.
Wash them twice with PBS and transfer 400 microliters of cells to each. Well finally change the medium after the first 24 hours, and then every other day until the experimental endpoint to remove any unattached dead cells. As illustrated in these images, replated single cell human pluripotent stem cell cultures will expand as clusters during the first 48 hours of differentiation, reaching about 70%confluence.
After differentiation, the cells achieve full confluence in as short as three to four days. By day 20, the differentiated cells attach and grow as small clumps. During the next five days, much more intensive and morphologically intact axons and neurites appear within the clumps, forming some connections between the clumps.
After 11 days of differentiation, the human pluripotent stem cells can be efficiently converted to floor plate precursors. As demonstrated in these images, almost all of the cells express the floor plate precursor, cell markers nest in and fox A two and over. 90%of the cells express Corin O OTX two and LM X one A.After further differentiation, the floor plate precursor cells specialize in to dopaminergic neurons that express TUJ one and TH.These dopaminergic neurons are of a nine identity as they express GK two and are negative for calbindin Immuno staining experiments also demonstrate that there are no GFAP astrocytes and very few GABAergic neurons indicating that the differentiation is exclusively specific for the dopaminergic neuron lineage.
While testing this procedure, it's important to keep consistency in the everyday cell culture and to make sure the medium is freshly prepared with correct ingredients.
