This video demonstrates a method for differentiation of primary neural human fetal progenitor cells into oligodendrocytes. First neural progenitor cells are cultured in progenitor medium to near co fluency C.The progenitor medium is then exchanged for oligodendrocyte medium, supplemented with the growth factors which promote the differentiation process. The cells are then cultured and expanded for three weeks.
The growth factor supplemented oligodendrocyte medium is then exchanged for oligodendrocyte medium without growth factors, and the cells are cultured for an additional six to 10 days until phenotypic changes are seen by microscopy. Differentiation is then confirmed by immunofluorescence microscopy and flow cytometry using oligodendrocyte lineage specific antibodies. Ultimately, the successfully differentiated oligodendrocytes can be used for in vitro studies of oligodendrocyte biology and interactions with infectious agents such as the human polyomavirus, JCV.
One of the real advantages of the technique we've established in this laboratory compared with what's already in the published literature is that in our system, the oligodendrocytes can be cultured and differentiated without the need of supporting cells such as astrocytes and neurons. Another advantage of our protocol, we think, is that it makes it possible to cultivate a large number of these precursor oligodendrocytes that express markers such as oh four that can be grown in pathogen culture for many weeks into large numbers of populations of cells while maintaining their phenotype. The end result of differentiation gives us almost a pure population of oligodendrocytes.
We first said the idea for this method when we read the extensive literature on the in vitro differentiation of oligodendrocytes in rat and my cells. Also different investigator were removing growth factors from narrow cultures in order to complete the differentiation of the cells. Demonstrating this procedure with our protocol will be Alexa Bandon, an undergraduate student in our laboratory here in the N-I-N-D-S.
In preparation for differentiation, grow a multipotential population of neural progenitor cells on polylysine coated flasks in neural basal medium, supplemented with BFGF and EGF, discard any unused cells. After 11, when the culture is approximately 70 to 80%confluent, initiate differentiation. To do this, aspirate the progenitor medium from the flasks.
Rinse the cells once with PBS and then add 12 milliliters of oligo medium with growth factors. Incubate the cells at 37 degrees Celsius in a humidified 5%carbon dioxide incubator. After one week, the remaining cells will exhibit a narrow bipolar morphology.
Once the differentiated cells are at least 70%confluent, remove the medium and transfer it to a fresh 50 milliliter conical tube. Set the saved conditioned medium aside for later use. Then quickly add four milliliters of 0.05%trypsin, 0.1%EDTA to the cells, and incubate them for five minutes at room temperature.
Gently tapping the flask several times to help cell detachment. After the incubation, quench the trips in by adding four to five milliliters of the saved conditioned medium to the flask. Next, transfer the medium and the cells back to the 50 milliliter conical tube.
Then centrifuge the medium and the cells at 310 RCF for seven minutes of room temperature. Once the centrifugation is complete, aspirate the ate. Re suspend the cell pellet by gently tapping the tube and then add fresh oligo medium with growth factors.
Gently pipette medium and cells up and down to make a uniform cell suspension. Count the cells using a hemo cytometer. Then transfer 2.5 million cells into new poly de lysine coated T 75 flasks, and place the culture in the incubator after three weeks when the cells are 80 to 85%confluent aspirate the oligo medium with growth factors from the flask.
Rinse once with PBS and then add 12 milliliters of oligo medium without growth factors and four milliliters of progenitor derived neuron unconditioned medium. Maintain the cells in this medium for six to 10 days on alternating days. Examine the cells by phase contrast microscopy to assess differentiation and co fluency and replace at least 50%of the medium after withdrawal from growth factors, the cells will progressively develop multiple processes indicating successful differentiation of the cells into progenitor derived oligodendrocytes.
At this point, the progenitor derived oligodendrocytes can be used for flow cytometry or immunofluorescence experiments as described in the upcoming sections of this video. The flow cytometry assay compares the acquisition of oligodendrocyte markers during the differentiation process in relationship with those of their parental population. Human neuro progenitor cells are used as controls C 2.5 million progenitor derived oligodendrocyte cells in each of two T 75 flasks coated with poly de lycine in oligo medium with growth factors, incubator 37 degrees Celsius a week later, replace the medium in one flask with fresh oligo medium containing growth factors as a control for cell viability.
Replace the medium in the second flask with fresh oligo medium without growth factors. Culture these cells for up to 10 days, three to nine days after the removal of growth factors, the cells will begin to acquire more processes typical of an oligodendrocyte phenotype. To dissociate the progenitor derived oligodendrocyte cells, aspirate the medium and add 5.25 milliliters of perian solution to each flask.
Perian is used instead of trypsin because it has a more gentle effect on the cells during the dissociation. Preserving the cellular morphology incubate at 37 degrees Celsius for 15 to 30 minutes. Periodically check the detachment of the cells using a microscope.
Once the cells are detached, add five milliliters of medium width or without growth factors to stop the reaction. Next, transfer the contents of each flask to a 15 milliliter conical tube. Centrif used the tubes at 310 RCF for seven minutes of room temperature following the centrifugation, aspirate the supinate and re suspend the cell pellets in four milliliters of normal physiological medium, supplemented with one milligram per milliliter, bovine serum albumin or NPM plus BSA.
Then count the cells as before, and then for each immunostaining condition, Eloqua 1 million cells into a five milliliter polypropylene tube. Be sure to include all the appropriate controls to confirm the specificity of each immuno reagent. Next to perform immuno staining, the cells are first incubated with antibodies against the surface proteins, A two B 5, 0 4, and gal C.The cells are then washed to remove unbound primary antibodies and incubated with the appropriate fluorochrome labeled secondary antibodies.
The cells are then washed again to remove the secondary antibodies. Fixed with 2%paraldehyde and permeated with cold, 70%ethanol then to stain for intracellular proteins, the cells are washed, then incubated with antibodies against nesting, glial fibrillary, acidic protein, class three beta tubulin and myelin basic protein. After the unbound, primary antibodies are washed away.
The cells are incubated with the appropriate fluorochrome labeled secondary antibodies. Finally, to discriminate between intact cells and subcellular a debris during flow cytometry analysis, the cells sustained with four six DIA medino. Two phenol Lind dihydrochloride or dappy perform flow cytometry on an instrument equipped with three lasers, which provide excitation wavelengths tuned to 4 88 nanometers, 6 47 nanometers, and a broad UV.In preparation for immunofluorescence microscopy, prepare six poly D lysine coated.
Six well plates with 250, 000 cells of progenitor derived oligodendrocytes per well. Two plates are needed for each time point. One cultured with growth factors and one without when the cells are 70 to 80%confluent.
Aspirate the medium with growth factors from the plate. Rinse once with PBS and then add 12 milliliters of medium without growth factors. Perform immuno staining to five and nine days after the withdrawal of growth factors as follows, first, fix the cells with 2%PFA for 10 minutes at room temperature.
Next, discard the PFA and wash the cells three times with PBS five minutes each time. Then the cells with 0.25%triton then to prevent non-specific binding block cells with 2%horse serum and 10%goat serum. After blocking, incubate the cells with antibodies against beta three tubulin, G-F-A-P-M-V-P oh four, and gal C.Then wash the unbound antibodies from the cells and incubate the cells with the appropriate fluorochrome labeled secondary antibodies.
Wash the unbound secondary antibodies from the cells and then to avoid photobleaching and distain. The nucleus mount. The cover slip using 10 microliters of prolonged gold with dpi.
Visualize labeled cells using a fluorescence microscope with the appropriate filter set to determine whether human, primary neural progenitor cells could differentiate into oligodendrocytes. Multi potent neural progenitor cells were cultured as described in this video. Phenotypic changes were then monitored by flow cytometry and immunofluorescence microscopy.
As can be seen here. The majority of neural progenitors express the neuro epithelial stem cell marker nesting, whereas only small subpopulations express lineage restrictive markers such as the astrocyte marker, GFAP, the neuronal marker, class three beta tubulin, or the oligodendrocyte marker oh four. When culture medium was replaced with oligo medium plus growth factors and cells were cultured for one week, decreased nesting expression and increased A two B five expression was observed after two weeks.
Nesting expression was decreased even further and expression of the neuroglial precursor marker, A two B five increased the expression of another oligodendrocyte marker oh four also increased two days post growth factor withdrawal. Oh four expression and expression of GAL C.A late oligodendrocyte marker increased six days after growth factor withdrawal. The co-expression of oh four and gal C increased and was comparable to the co-expression of gal C and MBP.
The multi epitope immuno staining reveals that 72%of the cells in culture expressed MBP. Further evidence of progenitor derived oligodendrocyte differentiation was characterized using an immunofluorescence assay. Two days after withdrawal of growth factors, the expression of MVP shown in green was detected.
This was increased at day five and further increased at day nine. The localization of MVP in the differentiated oligodendrocytes is evident. Taken together, these data indicate that human fetal neural progenitor cells cultures using the method described are able to proliferate in vitro while maintaining the capacity to differentiate through stages of the oligodendrocyte lineages.
Following the development of this protocol, the technique actually allows researchers in the fields of developmental biology, neuroscience virology to investigate the biology of the human brain and diseases that affect the human brain, and particularly those that result in white matter Diseases.
