The overall goal of this procedure is to generate human IPS cells via retrovirus mediated overexpression of OCT four, SOX 2K LF four, and M with a GFP reporter system. This is accomplished by first adding the appropriate amount of individual GFP expressing retrovirus. Next, the GFP expressing virus infected cells are transferred into 0.1%gelatin coated plates with mouse embryonic fibroblasts.
The third step of the procedure is to replace the medium with human embryonic stem cell culture medium. Finally, GFP silenced IPS colonies that show a similar morphology to human embryonic stem cells are isolated. Ultimately, the results show the expression of endogenous pluripotent markers through immunofluorescent staining and R-T-Q-P-C-R.
The main advantage of this technique of existing method like lifestyle staining with pluripotent surface marker and observation of a morphological change is the desirable iPad. Cell colonies can be easily isolated using loss of GFP expression as a marker for the pluripotent cells To begin culture. Human fibroblasts in fibroblasts medium one day before infection plate one times 10 to the fifth human fibroblasts into one well of a six well plate the next day aspirate the medium to remove dead cells and add two milliliters of fresh fibroblast medium with five micrograms per milliliter of protamine sulfate.
Then carefully add the appropriate amount of each GFP expressing virus corresponding to a multiplicity of infection of five. Incubate the cells at 37 degrees Celsius overnight one day after infection. Remove the viral supernatant wash three times with two milliliters PBS.
Then add two milliliters, fibroblast medium three days after infection. Check the GFP fluorescence and replenish the well with two milliliters. Fibroblast medium the following day plate one times 10 to the fourth per square centimeter of irradiated mouse, embryonic fibroblast or meth feeder cells in fibroblast medium onto a 10 centimeter Petri dish coated with 0.1%Gelatin.
Incubate the cells at 37 degrees Celsius overnight. The next day, detach the infected human fibroblasts with one milliliter of 0.05%tripsin EDTA for five minutes at 37 degrees Celsius and centrifuge for five minutes at 200 G.Resuspend the cells in 10 milliliters of fibroblast medium and replay the cells on pre-coated 0.1%gelatin with mfs. 24 hours later.
Replace the medium with HESC culture medium and change the medium daily. ESC like colonies will start to appear 20 to 27 days after infection after the appearance of ESC like colonies under a fluorescence microscope. Check for the absence of GFP fluorescence in a colony that shows similar morphology to HSCs.
Using a 10 microliter pipette. Pick individual IPSC colonies and place them into one well of a gelatin and meth coated 12 well plate and supplemented with HESC medium. Change medium daily passage the cells by washing the plate with one milliliter of DMM F 12.
Then add 0.5 milliliter of collagenase and incubate for 10 minutes at 37 degrees Celsius. Wash the cells twice with DM A MF 12. Next, add two milliliters of fresh HESC medium.
Then use a cell lifter to break up the colonies into small pieces and detach the remaining cells from the plate. Transfer the resuspended pieces of colonies into one well of a gelatin and MEF coated six. Well plate and incubate at 37 degrees Celsius for two days.
To check the cells by immunofluorescence. Wash them three times with PBS and fix them with 4%paraldehyde for 20 minutes at room temperature. Gently wash the cells three times with PBS and perme them with 0.2%tritton X 100 in PBS for 30 minutes.
Block non-specific binding by incubating the cells with 3%BSA in PBS for two hours. Next, incubate the cells with primary antibody overnight at four degrees Celsius. Then wash the cells three times with PBS and incubate them with secondary antibody for one hour at room temperature in the dark.
Wash the cells three times with PBS during the last wash. Add dappy and incubate at room temperature for five minutes. Image the cells using a fluorescent microscope for quantitative realtime.
PCR isolate total RNA from human IPCs derived from human fibroblasts. Using an RNA isolation kit. Synthesize first strand cDNA using reverse transcriptase.
Then use it as the template for QPCR to detect pluripotency genes. Human fibroblasts, BJ one that were infected with a cocktail of retroviruses carrying OCT four, SOX two, KLF four, and Mick shown here are the morphological changes observed during reprogramming from day five. Today's 10, 14 and 21, the cells begin to show the HESC like morphology after 21 days, and the IPCs are recognized by GFP fluorescence.
Pluripotent stem cells such as ESCs and IPCs express the molecular machinery to repress proviral gene expression. However, the retroviral vectors used here express GFP together with reprogramming genes by retroviral LTR. Thus, cells continuously expressing GFP are considered to express transgenes without proviral gene silencing as seen here faithfully, reprogrammed IPSC colonies that acquire the Pluripotency Molecular Network show the absence of GFP expression.
This figure shows immunohistochemistry analysis of colonies derived from Detroit 5 51 fibroblasts with antibodies to TRA 180 1, TRA one 60 SSEA four T four, and nano as can be seen successfully. Reprogrammed IPCs express all of these markers. In addition, quantitative R-T-P-C-R was carried out to analyze gene expression and revealed that T four SOX two, KLF four M and nano were significantly increased compared to the parental fibroblast cells, but commensurate with that of H nine HSCs.
After watching this video, you should have a good understanding of how to infect the virus, identify and pick up the fully lip programmed IPA cells using fluorescent microscopy.
