The overall aim of this experiment is to achieve high transfection efficiency in human-induced pluripotent stem cells using optimized transfection and nuclear affection protocols. This is accomplished by first establishing feeder free cultures of human-induced pluripotent stem cells. For transfection, the human-induced pluripotent stem cells are plad on gelt, TRX, or matrigel overnight.
While for nuclear affection, small cell clumps are obtained by trypsin, a plasmid DNA and gene juice transfection reagent mixture is added to the plated cells and then the plate is spun and incubated at 37 degrees Celsius. The human IPS cell suspension is nuclear affected in a nuclear affected device, and cells are resuspended in meth conditioned, medium, and incubated at 37 degrees Celsius. Ultimately, results can be obtained that show efficient genetic manipulation through high transfection efficiency in human IPS cells.
The main advantage of this technique over existing methods like previously described transfection electroporation and nuclear affection methods is that high transfection efficiency in human IPS cells with consistent outcomes can be routinely achieved in our lab. Our protocols also ensure minimal cell death following genetic modification. Hence, our protocols permit a rapid and large scale generation of transgenic human IPS cell lines Prior to adapting human induced pluripotent stem cells to feeder free cultures prepare gelt TRX coated dishes.
Next, massage The human IPCs previously maintained on feeder cells at one milliliter of Accutane per well and incubate at 37 degrees Celsius for one minute and until most cells start to detach, add 10 to 15 glass beads to the cells and gently swell the plate. Then add two milliliters of knockout D-M-E-M-F 12 and gently tritrate. Transfer the cell suspension to a 10 milliliter centrifuge tube.
Spin the cells at 800 RPM for three minutes at room temperature. Aspirate the supinate, leaving the human IPSC pellet intact. Gently flick the tube to disperse the cell pellet gently.
Reese, suspend the human IPSC pellet in an appropriate volume of STEM pro medium distribute between the wells of a gel TRE coated plate. Depending on the proliferation rate, human IPCs can be passaged in a split ratio of one to two, to one to six. Carefully place the plate into a 5%carbon dioxide, 37 degrees Celsius incubator and swir the plate gently to ensure an even distribution of cells across the wells.
Feed cells daily. When cells reach 80%co fluency, they're ready to be passaged again onto a new gel. Rex coated well at a split ratio of one to two cells should be approximately 40 to 50%confluence and in small colonies distributed evenly on the gel coated well on the day of transfection to achieve optimal transfection efficiency.
To begin this procedure, prepare 100 microliters of knockout medium in a sterile 1.5 milliliter eend orph tube. Add 27 microliters of gene juice transfection reagent, mix well and incubate it room temperature for five minutes. Next, add four micrograms of plasmid DNA.
The choice of plasmid is critical for optimal transfection efficiency. The plasmid used here has an enhanced green fluorescent protein driven by a CAG promoter, which is a strong promoter that is transcriptionally active in human IPCs. Incubate the tube room temperature for 15 minutes.
After 15 minutes, add the gene juice, DNA mixture to the cells and swell the plate. Spin the plate at 1, 200 RPM for five minutes to increase the contact of transfection mixture with human IPCs in the well. Incubate the cells at 37 degrees Celsius overnight on the following day.
Monitor for transfection efficiency on the day of nuclear affection pretreat human IPCs with 10 micromolar rock inhibitor for at least one hour of 37 degrees Celsius while the human IPS CS are being pretreated. Prepare 82 microliters of human stem cell nuclear effector solution in a sterile 1.5 milliliter eend orph tube. Add 80 microliters.
Supplement one and mix well by pipetting. Incubate the solution at 37 degrees Celsius for five minutes. Pre-war, previously prepared feeder cell conditioned media and 0.25%trips in EDTA to 37 degrees Celsius under the hood.
Pre label one sterile 15 milliliter conical tube. Carefully remove the media from the human IPSC culture to be nuclear affected and gently wash the cells with phosphate buffered saline. Aspirate the PBS and add trypsin incubate cells at 37 degrees Celsius for three minutes.
For successful nucle affection, it is critical not to trypsin the cells into single cells. Instead, the cells should be triturated into small clumps consisting of two to three cells. Gently rerate the cells with a P 1000 pipette tip into small clumps consisting of two to three cells.
Wash the bottom of the well by repeated pipetting to make sure all human IPCs are completely detached. Then transfer the cell suspension to the labeled 15 milliliter conical tube. Next, add nine milliliters of mouse embryonic fibroblasts media.
To inactivate the tripsin, spin the cells at 800 RPM for three minutes. Carefully aspirate the SUP natant leaving the cell pellet intact res. Suspend the cells in 100 microliters, a pre-war human stem cell nuclear effector solution.
Using a one milli milliliter pipette tip transfer cells to a nuclear effector vete. Add four micrograms of plasma DNA into the cell suspension in the vete mix cells and DNA by gentle swirling. Tap the vete twice on the hood surface.
Insert the vete into the nuclear effector holder of the nuclear effector device. Select program B 0 1 6 nuclear effect cells by pressing button x Okay will be displayed When the nuclear affection process is completed, which usually takes one to five seconds. Use the plastic pasta pipette provided in the nuclear affection kit to retrieve nuclear affected cells from the vete recover cells by Resus, suspending them in prewarm CM and rock inhibitor in a sterile 1.5 milliliter eend orph incubate cells at 37 degrees Celsius for 10 minutes.
After 10 minutes, use a one milliliter pipette tip to transfer the cells dropwise onto feeder layers. Incubate the cells at 37 degrees Celsius overnight on the following day. Monitor for transfection efficiency.
Shown here are photo micrographs of EGFP expressing RIV one human IPSC cells transfected using gene juice on gel Rex 12 hours post transfection plasmid. DNA transfection of human IPCs. Using gene juice with a strong promoter typically yields more than 60%of EGFP reporter expressing cells in a transient transfection assay.
EGFP expressing cells can also be obtained by nuclear affection as illustrated in these images of RIV one human IPCs plated and formed on feeders following nuclear affection using either the B 0 1 6 nuclear affection program or the A 0 2 3 nuclear affection program. The use of the B 0 1 6 program results in up to 80%of EGFP positive cells, but the A 0 2 3 program yielded less than 10%of EEG FP positive cells. Furthermore, stable clones of transgenic human I PSCs that express the trends gene can be derived.
An example of stable EGFP expressing human IPSC colonies with ubiquitous EGFP expression derived from gene juice is shown here. These cells retain constitutive EGFP expression during embryo body differentiation. After watching this video, you should have a good understanding of how to nucle effect and transfect human IPS cells.
Efficient genetic manipulation is an essential tool to study various aspects of stem cell biology and will accelerate the use of human IPS cell lines in regenerative medicine.
