The overall goal of the following experiment is to convert cells derived from human brain parasites into induced neurons. This is achieved by isolating and culturing parasite derived cells from surgical brain specimens. Next, following expansion, in vitro cells are immuno labeled for parasitic markers, which allows for the characterization and eventual further enrichment of the cells.
Then parasite derived cells are transduced with retroviral vectors encoding neuronal fate determinants in order to directly convert them into induced neurons. Results are obtained that show induced neurons obtained from lineage reprogrammed parasite derived cells based on immuno staining for the neuronal marker beta three tubulin. The main advantage of this approach over existing methods like reprogramming of fibroblasts or differentiation of embryonic stem cells into neurons is that we target cells endogenous to the brain, which may ultimately pave the way for direct lineage conversion within the brain without having to transplant the cells.
The following protocol was developed in accordance with the approval by the ethical Committee of the medical faculty of the LMU Munich and written informed consent from all patients beginning with a fresh human brain biopsy. Keep on ice in HBSS and heaps dissociate the tissue into single cells by transferring it into a 65 millimeter Petri dish and using two sterile single-use scalpels, mince it into small pieces. Transfer the tissue to a 15 milliliter conical tube and add three to six milliliters of trip le incubate for 15 to 30 minutes at 37 degrees Celsius in a water bath.
After the digestion reaction, add one volume of prewarm growth medium to facilitate the association and use a five milliliter disposable pipette followed by a glass pasture pipette to gently T tri rate the cell suspension. Spin down the solution at 157 Gs for five minutes. Re suspend the pellet in growth medium and transfer the culture to a T 75 flask.
Incubate the cells at 37 degrees Celsius with 5%carbon dioxide and 5%oxygen. Replace half of the medium every three to four days until cells have reached co fluency. When the cells have reached co fluency, aspirate the culture medium and wash with room temperature PBS before adding three milliliters of trip le.
Following a five to 10 minute incubation at 37 degrees Celsius. Gently tap the flask to help lift the cells. Then add three volumes of growth medium after spinning down the suspension and resus suspending the cells in growth medium, passage them at a one to three ratio for optimal yield into new T 75 culture flasks.
The cells have been passaged up to five times without any signs of reduced reprogramming and endothelial cell numbers will become negligible with each passage to carry out immuno cyto chemistry seed approximately 60, 000 cells onto poly D lysine. Coated glass cover slips in 500 microliters of fresh growth medium in 24. Well tissue culture plates and culture.
The cells for 24 hours at 37 degrees Celsius with 5%carbon dioxide and 5%oxygen. For immunochemical analysis of the cultured human brain cells, remove the medium and wash three times with one milliliter of PBS. Add 500 microliters of 4%paraldehyde in PBS and incubate for 15 minutes.
At room temperature. Transfer the glass cover slips into an appropriate humidified staining chamber and add 80 microliters of P-B-S-B-S-A Triton X 100 block for one hour at room temperature before adding primary antibodies diluted in the same solution and incubating one hour at room temperature or overnight at four degrees Celsius. Use PBS to wash the cells three times before adding secondary antibodies and incubating for one hour at room temperature.
After washing three times, apply anti fading medium and mount the cover slips. Use epi fluorescence or confocal microscopy to analyze the samples. Adhere to local biosafety guidelines when handling retroviral particles 24 hours prior to retroviral transduction seed, approximately 60, 000 cells on top.
Poly de lysine coated glass cover slips in 500 microliters of fresh growth medium in 24. Well tissue culture plates and incubate at 37 degrees Celsius. The next day.
Replace the growth medium with 500 microliters of fresh prewarm growth medium and add one microliter of the respective concentrated supernatants containing retroviral particles. Gently shake plate to ensure even viral particle distribution throughout the well. One day later, remove the medium with viral particles from the cells and add one milliliter of fresh prewarm.
B 27 differentiation medium as outlined in the text protocol, maintain the cells in culture for four to eight weeks without changing the medium as this can harm induced neurons as they mature to demonstrate the acquisition of a neuronal phenotype. Perform immuno cyto chemistry against neuronal antigens such as beta three tubulin map two and nen. As demonstrated earlier in this video, immuno cyto chemistry performed on successfully established cultures from human adult cerebral cortex reveals a considerable degree of heterogeneity between cultures derived from specimens of different patients as quantified by flow cytometry.
There is a substantial fraction of cells that express P DG FR beta and other parasite markers such as CD 1 46, NG two, and CD 13 are expressed at lower levels. Likewise, SMA is expressed in a minor subpopulation of cultured cells at passage zero, there are usually less than 10%of CD 34 positive endothelial cells and they decline below detection with further packaging. Likewise, astrocytes comprise only a negligible contamination at earlier passages.
Immuno phytochemical and flow cytometry data are fully corroborated by quantitative R-T-P-C-R-A. Further enrichment of parasite derived cells can be achieved by facts at this stage. This is particularly relevant when the purity of the culture is at the lower end of the spectrum as reflected by the level of P-D-G-F-R beta expression.
Thus, we have utilized an antibody against P-D-G-F-R beta CD one 40 B alone or in combination with an anti CD 1 46 antibody while excluding any CD 34 positive contaminants to facts sort. Specifically, parasites transducing these cultures with control viruses does not alter their expression profile indicating that they remain in the parasite lineage. Likewise, retrovirus mediated expression of SOX two alone does not result in any changes in morphology nor does it induce beta three tubulin expression.
In contrast, approximately 10%of the cells transduced with an A SCL one encoding retrovirus alone. Exhibit beta three tubulin expression, but don't acquire a neuronal morphology strikingly nearly 50%of all, so two and a SCL one cot. Transduced cells show beta three tubulin expression, and a third of them also display neuronal processes indicating their fate conversion into PNS For following this procedure.
Other techniques like patch ggl electrophysiology can be employed in order to address additional questions regarding the maturation and functionality of induced neurons.
