The overall goal of this procedure is to shorten the classical Yamanaka IPS approach by directly generating induced neural progenitor cells from a patient's skin biopsy for therapeutic applications. This is accomplished by first performing a punch biopsy from the patient to derive primary fibroblast cells that can be cultured in vitro. The second step is to expand and subsequently infect cultured fibroblasts with viruses that encode reprogramming factors to induce trans differentiation.
Next, the putative converted induced neural progenitor cell or INPC colonies must be carefully selected by visual validation and subsequently isolated by manual picking approximately 20 days after infection. The final step is to monoclonal, expand the IPCs in neuro induction medium, ultimately targeted in vitro differentiation as well as immunofluorescence microscopy is used to show that patients own directly converted IPCs are able to differentiate into neuronal as well as glial cells making them a virtually unlimited source for biomedical applications. The advantage of this direct conversion technique over existing methods like the Yamanaka type derivation of induced blue potent stem cells and their subsequent differentiation is twofold.
DI conversion into multipotent induced neuro stem cells, we call them INS cells, is not only considerably faster but also safer. Thus, the trans differentiation procedure will make it much more achievable to produce patient specific cells for therapeutic applications and harvest a dramatically lower risk of tumor formation in comparison to IPS cells. By that, we are now able to dismantle two major roadblocks of region rate of medicine nowadays.
That's clinical safety and efficiency. To begin the punch biopsy disinfect the skin of the patient, anesthetize the skin where the biopsy will be taken with 0.5 to one milliliter of mepivacaine hydrochloride intra cutaneous. Take the skin biopsy using a sterile three millimeter biopsy punch.
Rinse the biopsy with dobe echo's phosphate buffered saline or DPBS plus one microliter per milliliter of gentamycin. Remove the fat from the biopsy using a scalpel and forceps. Then rinse the biopsy twice more with the buffer before aspirating it completely.
Cover the biopsy with dis paste two and incubate for 16 to 18 hours at four degrees Celsius. Following incubation, aspirate the dys paste completely before washing the biopsy twice with DPBS. Remove the epidermis with tweezers, then wash the biopsy twice more with DPBS.
Next, add two milliliters of collagenase type two and transfer to a 15 milliliter tube in the tube. Add collagenase up to five milliliters. Total volume incubate for 45 minutes at 37 degrees Celsius.
Centrifuge the sample for five minutes at 180 times G and discard the snat afterwards. Resuspend the pellet in 10 milliliters of DMEM fetal calf serum gentamicin medium before centrifuging again for five minutes at 180 times G.Discard the supernatant and resuspend the pellet in 1.5 milliliters of the same medium. Then transfer the sample to a T 25 adherent tissue culture flask and incubate at 37 degrees Celsius 5%CO2, changing the medium every three days after approximately two weeks.
Once the cells are confluent around the skin parts, split the cells using trypsin EDTA as described in the text protocol. Further expand the cells by changing the medium every third day and splitting when cells are confluent. After excluding mycoplasma contamination by standard assays, prepare the cells for the direct conversion experiment by washing the cells once with DPPS then aspirate the DPBS and add 2.5 milliliters of trypsin EDTA After incubating the cells for five minutes at 37 degrees Celsius, 5%CO2, tap the flask gently to detach the cells.
Resuspend the cells in 2.5 milliliters of fibroblast medium and transfer to a 15 milliliter tube. Centrifuge the tube at 180 times G for five minutes and aspirate the snat. Resuspend the cells in one milliliter of fibroblast medium and pipette up and down to generate a single cell suspension.
Count the number of cells using a cell counting chamber before plating. 30, 000 cells per well. In a 24 well plate incubate the cells overnight at 37 degrees Celsius, 5%CO2 steps handling virus must be performed in a biological safety cabinet with appropriate personal safety equipment, including a surgical mask to prevent mucosal exposure.
To perform the infection first, replace the existing cell medium with 100 microliters of fibroblast medium. Then resus resuspend thawed aliquots of T four, KLF four, SOX two, and cmix sendi virus In one milliliter of fibroblast medium. Add each virus in an MOI of three to the cells and mix gently incubate the cells overnight at 37 degrees Celsius, 5%CO2 after 24 hours, aspirate the medium and add 500 microliters of neuro induction medium culture, the cells at 39 degrees Celsius, 5%CO2 from now on, changing the medium every other day on day six after infection, prepare laminin coated six well plates.
Add one milliliter of one microgram per milliliter laminin in DPBS onto six well plates and keep the plates at four degrees Celsius for 24 hours on day seven after infection, split the cells using D-P-B-S-E-D-T-A as described in the text protocol. Add 500 microliters of D-M-E-M-F 12 and transfer the suspension to a 15 milliliter tube. Then centrifuge the sample at 180 times G for five minutes.
Aspirate the supernatant and resuspend the pellet in 1.5 milliliters of neuro induction medium plate the cells on laminate coated six well plates and add RO kinase inhibitor to a final concentration of 10 micromolar culture. The cells at 39 degrees Celsius, 5%CO2, changing the medium every other day from day 14. After infection culture, the cells at 37 degrees Celsius 5%CO2 neuro epithelial colonies become apparent around day 17 after infection as visualized by phase contrast microscopy.
They should be large enough to be picked around day 20 after infection one day before picking coat 1 48. Well plate with laminin as described in the text protocol one day later, aspirate laminate from the plates and add 200 microliters of neuro induction medium to the wells. Wash the six well plates containing the colonies to be picked once with DPBS before adding two milliliters of neuro induction.
Medium per well of a six Well plate mechanically pick the cells by scraping around the colonies using a thin needle to get rid of surrounding cells and picking the colonies using pipette tips. Mounted on a 200 microliter pipette man set on 50 microliters. Transfer each colony into one well of a laminate coated 48 well plate and generate a single cell suspension mechanically by pipetting up and down 10 times.
Add R kinase inhibitor in a final concentration of 10 micromolar to the cells. Grow the cells on the 48 well plate at 37 degrees Celsius, 5%CO2 for two days. Change the medium every second day until the cells reach 80 to 90%co fluency.
See the text protocol for expansion and cryo-preservation of the IPCs for differentiation of IPCs towards a neuronal lineage culture. The cells on laminate coated plates. As before, change the medium to neuro diff medium when the cells are approximately 70%confluent and continue to change the medium every other day for three weeks.
Do not split the cells during differentiation after three weeks, cells should express the neuronal marker TUJ one to gain more mature neurons and neuronal subtypes. Continue differentiation up to three months for differentiation of IPCs, specifically towards a glial lineage. Change the medium to glial induction medium, including 20 nanograms per milliliter of epidermal growth factor to induce differentiation into glial precursor cells.
Remove half of the medium and replace with fresh medium every other day for about two weeks. During this period, cells should be split one to three in the presence of 10 micromolar R kinase inhibitor when 100%co fluency is reached after two weeks. Differentiate the cells further into astrocytes by changing the medium to commercially available astrocyte medium when the cells are nearly confluent, and by continuing to change the medium every second day, approximately seven days later, cells start to express astrocyte markers.
If cells get 100%confluent during the differentiation protocol, split them in a one to two ratio. In the presence of 10 Micromolar RO kinase inhibitor infection of the fibroblasts with T 4K LF four, SOX two, and cmix sendi viruses and culture in neuro inductive medium conditions resulted in a change of morphology of the fibroblasts and subsequent emergence of colonies. 17 days after infection generated, monoclonal cell lines can be expanded and stain positive for neural stem cell markers such as SOX two, SOX one neston, and PAC six, as well as for proliferation marker KI 67, whereas they do not express the pluripotency associated marker T four.
Moreover, by addition of neuronal growth factors to the cell culture medium, the neural progenitor cells can be differentiated into neurons by applying an astrocyte differentiation. Protocol induced neural progenitor cell lines can also be differentiated into glial lineages as judged by analysis of typical morphological changes and staining against GFAP. By this direct conversion, we generate multipotent induced neuro progenitor cells from patient fibroblasts within three to four weeks.
These cells could be used for numerous biomedical applications. The implications of this technique extend towards therapy, diagnosis and modeling of neurodegenerative disorders like Alzheimer's and Parkinson's disease, as well as other neurological conditions.
