Due to its high efficiency, this method can further advance induced pluripotent stem cells, or IPSCs as a tool for studying human disease and developing novel stem cell therapies. The main advantage of this protocol is the ability to generate high-quality integration free IPSCs from primary human fibroblasts including difficult to reprogram, disease associated, aged, and senescent fibroblasts. The success of this method depends on optimal RNA transfection efficiency of fibroblasts.
And adjusting the pH of a transfection buffer is a key procedure to achieve this. To start, prepare transfection buffer by using 500 milliliters and 100 milliliters room temperature pre-warmed bottles of fresh reduced serum medium. Measure the base pH of the medium in 500 milliliter bottle by inserting the pH meter's glass electrode into the buffer, and wait up to one minute before reading the pH.
To adjust the pH, add three to four milliliters of one molar sodium hydroxide into 500 milliliter bottle. Close the bottle and mix well. After waiting for five minutes, open the bottle and insert the pH meter's electrode into the buffer.
Wait until the reading on the pH meter stabilizes. Continue adding small volumes of one molar sodium hydroxide until the pH reaches 8.15 to 8.17, making sure to calibrate the pH meter several times during the process. Use a 0.22 micrometer vacuum filtration system to filter sterilize the transfection buffer.
Aliquot the sterilized buffer into five milliliter tubes with minimal air space. Verify that the fibroblasts to be reprogrammed are at 40%to 60%confluency. Transfer four milliliters of DPBS into a 15 milliliter conical tube, and add 100 microliters of recombinant human laminin 521.
Mix thoroughly by pipetting up and down. Add one milliliter per well of diluted recombinant human laminin 521 into three wells of a six well plate. Incubate this coated plate at 37 degrees celsius for two hours.
During this incubation, warm six milliliters of fibroblast expansion medium and four milliliters of plating medium to 37 degrees celsius. Supplement the plating medium with basic FGF at a final concentration of 100 nanograms per milliliter and B18R at a final concentration of 200 nanograms per milliliter. Carefully aspirate the spent medium from fibroblasts that are at 40%to 60%confluency.
Rinse the cells with five milliliters of DPBS. Add three milliliters of trypsin EDTA and gently rock the plate to cover the cells with trypsin EDTA. Aspirate excess fluid, leaving approximately 500 microliters of trypsin, and incubate the fibroblasts for three minutes at 37 degrees celsius.
After removing the plate from the incubator, firmly but carefully tap the side of the plate to dislodge the cells. View the cells under the microscope to check whether they have been detached. Add five milliliters of fibroblast expansion medium to the detached cells to neutralize the trypsin EDTA.
Collect the cells in a 15 milliliter conical tube and mix them well. Count the cells using a hemocytometer, then pipette 12, 000 cells into four milliliters of previously prepared pre-warmed plating medium. After removing the coated plate from the incubator, aspirate diluted laminin 521 from the wells, making sure that the surface of the wells doesn't dry.
Gently resuspend the cells that are in the plating medium and add one milliliter of cell suspension into each coated well. Place the plated cells into a tri-gas tissue culture incubator with low oxygen or oxygen set to 5%And then gently but thoroughly disperse the cells by alternating between an up down, then left right motion, and repeat the motions two more times, making sure not to swirl the plate. Incubate the cells overnight.
Meanwhile, add four milliliters of reprogramming medium to a 15 milliliter conical tube, and place it in the low O two incubator with a loosened cap to equilibrate overnight. At least one hour prior to transfection, remove the equilibrated reprogramming medium from a low O two incubator. Add bFGF to a final concentration of 100 nanograms per milliliter, and B18R to a final concentration of 200 nanograms per milliliter, and mix well.
Working one well at a time, use a one milliliter pipette to remove the spent medium. And replace it with one milliliter of reprogramming medium, supplemented with bFGF and B18R. Move the plate with cells back into the low oxygen incubator.
To start fibroblast transfection, first equilibrate an aliquot of the transfection buffer for approximately one hour at room temperature. Remove a single 33 microliter aliquot of modified MRNAs and 14 microliter aliquot of MIRNA mimics from negative 80 degrees celsius, and warm them to room temperature for approximately three to five minutes until thawed. Spin them down briefly in a microfuge.
Warm the transfection reagent to room temperature for approximately three to five minutes. Invert the tube two to three times to mix the reagent and spin it down briefly in a microfuge. Transfer 279 microliters of the room temperature transfection buffer into an RNAs free microcentrifuge tube, and add 31 microliters of the transfection reagent.
Mix thoroughly by pipetting and incubate at room temperature for one minute. Add 132 microliters of the room temperature transfection buffer to the 33 microliter aliquot of modified MRNA, and gently pipette to mix. Add 102.6 microliters of the room temperature transfection buffer to the 14 microliter aliquot of MIRNA mimics and gently pipette to mix.
To prepare the transfection mix of modified MRNA, add 165 microliters of the 310 microliters transfection buffer, transfection reagent mix to 165 microliters of the transfection buffer, modified MRNA mix. Pipette to mix. To prepare the transfection mix of MIRNA mimics, add 116.6 microliters of the 310 microliter transfection buffer, transfection reagent mix to the 116.6 microliter transfection buffer, MIRNA mimic mix.
Mix well and incubate for 15 minutes at room temperature to allow the transfection buffer to complex with the modified MRNAs and MIRNA mimics. Remove the plate with cells from the low oxygen incubator. Drop wise across each well, add 100 microliters per well of the transfection mix containing the complexed, modified MRNAs.
Gently but thoroughly agitate the plate with an up down, then left right motion to disperse the transfection complexes. Add 66.7 microliters per well of the transfection mix containing the complexed MIRNA mimics drop wise across each well. Gently but thoroughly agitate the plate with an up down, then left right motion to disperse the transfection complexes.
Place the plate with the transfected cells into the tri-gas incubator with low oxygen. Disperse the transfection complexes by gently but thoroughly agitating the plate with an up down, then left right motion. Incubate the transfected cells in the incubator for 16 to 20 hours before changing medium the following day.
Add four milliliters of reprogramming medium to a 15 milliliter conical tube, and place it in the low O two incubator with a loosened cap to equilibrate overnight. The following morning, replace the transfection containing medium with the fresh pre-equilibrated medium supplemented with bFGF and B18R and continue with the transfection regimen as described in the protocol. After successful plating into a well of a six well format dish for reprogramming, fibroblasts appeared very sparse.
24 hours following the first successful transfection, fibroblasts lost their spindle shape and adopted a more rounded morphology. Throughout the first three transfections, cell density gradually and consistently increased with an apparent burst in proliferation occurring between days seven and eight. By day 10, the cells appeared largely confluent.
The first IPSC colonies appeared as early as day 11. By days 15 to 17, IPSC colonies became large and obvious and clearly distinct from surrounding and completely reprogrammed fibroblasts. Immunostaining for the pluripotency marker TRA-1-60 confirmed high reprogramming efficiency.
Suboptimal plating density is the most common reason for reduced efficiency of reprogramming in this protocol. If plating density was too low, large acellular baron patches appeared and IPSC colonies did not form. Following dilution passage of reprogrammed cells, IPSCs grew as single colonies and were easily distinguishable from fibroblasts.
They were loosely packed and individual cells had a relatively large cytoplasmic area. Over the course of four to seven days, the IPSCs proliferated and formed a characteristic tightly packed colony with defined edges. Individual cells within the colony showed a large nuclear fraction with prominent nucleoli.
After reprogramming patient fibroblasts, the resulting IPSCs can be differentiated into a variety of somatic cells in order to elucidate disease causing mechanisms or develop novel regenerative cell-based therapeutics.
