The overall goal of these procedures is to visualize and track the progression of somatic cell reprogramming into induced pluripotent stem cells or iPSC. This method can be used to answer key questions in the iPSC field, such as how reprogramming kinetics and reprogramming efficiencies are altered by the somatic tissues used, the reprogramming technologies applied, and the matrix and media combinations used with it. The main advantage of this method is that it utilizes cell surface markers to look at the transitions of somatic cells into iPSCs such that you can use this technology to be able to monitor in real time the reprogramming events.
On day seven post transduction, wash the fibroblasts with two milliliters of DPBS per well and add 0.5 milliliters of 37 degree celsius 05%EDTA to each well for a three to five minute incubation at 37 degrees celsius. When the cells have rounded, stop the reaction with two milliliters of 37 degree celsius fibroblast medium per well and gently pipette the medium across the bottom of each well to dislodge the cells. Transfer the fibroblasts from each well into individual 15 milliliter conical tubes and collect them by centrifugation.
Resuspend the pellets in two milliliters of fresh 37 degree celsius fibroblast medium and count the cells from each culture condition. Next, seed one times 10 to the fifth cells into pre-prepared feeder-independent conditioned wells in a six-well tissue culture plate and incubate the transduced cells in the cell culture incubator overnight. The next day, replace the medium in each well with the appropriate medium for the cell application.
Then place the plate in the imager and set the software to capture continuous whole-well phase contrast and fluorescent images for each well every six to eight hours for the next 14 days. At the end of the reprogramming experiment, wash the wells one time with two milliliters of 37 degree celsius DMEM/F12 medium. Discard the wash and label the cells with one milliliter per well of the appropriate primary antibodies in fresh DMEM/F12 medium for 45 minutes at 37 degree celsius in the incubator.
At the end of the incubation, wash the cells two times with 37 degree celsius DMEM/F12 medium. Then discard the wash and label the cells with one milliliter of the appropriate secondary antibodies in 37 degree celsius DMEM/F12 medium for 30 minutes at 37 degree celsius. At the end of the incubation, wash the cells two more times with DMEM/F12 medium.
Then replace the wash in each well with two milliliters of fresh 37 degree celsius DMEM/F12 medium. Place the dish into the imager and open the imaging software. To set up scans on the imaging system software, first select the position of the tray to be used and select the type of dish to be scanned.
Next, select Whole Well Imaging, the pattern for scanning the desired wells, and the appropriate fluorescent channels for the real-time identification of the surface markers of interest. Then label the experiment to be scanned and set the time points and intervals for the samples to be scanned each day. To analyze a set of scanned experiments, select the experiment of interest and select Launch New Analysis Job.
Then select the appropriate processing definition for the type of analysis according to the channels used for the imaging, for example, PSC Phase Colony Count. Name the analysis job. And select the time range for the analysis and the wells to be analyzed.
Then launch the analysis job. To measure the desired reprogramming time points from individual wells, wash the well of interest one time with two milliliters of DPBS. Then replace the wash with one milliliter of pre-warmed 37 degree celsius 05%trypsin-EDTA solution.
After five minutes of 37 degree celsius, flush the trypsin against the bottom of the well to dissociate the cells into a single-cell suspension and transfer the cells into a 15 milliliter conical tube. Using three milliliters of DPBS, wash any of the remaining cells from the well and pull them in the 15 milliliter tube. Add an additional 10 milliliters of DPBS to the cells and spin them down by centrifugation.
Resuspend the pellet in one milliliter of DMEM/F12 medium for counting. Then dilute the cell suspension to a one times tenth of the six cells per milliliter concentration and aliquot one milliliter of cells per tube for the appropriate number of samples. Next, label the cells with the primary antibodies of interest in one milliliter of DMEM/F12 medium for 30 to 45 minutes at room temperature with mixing.
At the end of the incubation, wash the cells two times in 37 degree celsius DMEM/F12 medium. Resuspend the pellets in one milliliter of DPBS and transfer the samples into individual FACS tubes. Acquire the flow cytometry profiles of the reprogrammed plates.
Then transfer the FCS files to a computer and import the files into the appropriate analysis software. Open the first control file in a forward by side scatter plot and set the y-axis to a logarithmic scale. Then create a polygonal gate around the live population.
Next, create a side scatter area by scatter height plot with the live cells and set a rectangular gate to exclude the doublets. Then use these gates to analyze the experimental sample files under the appropriate fluorescent channels to identify the populations of interest in their respective quadrants. Reprogramming BJ fibroblasts with Sendai reprogramming viruses generates SSEA4+CD44-cells at a different rate than reprogramming DF1 fibroblasts, demonstrating that an early comparison of the percentage of pluripotent stem cell-like SSEA4+CD44-cells can be used to track the reprogramming progression.
In a flow cytometry time course, CD24-CD44+and EpCAM negative CD44+fibroblasts form double negative populations that later transition into CD24+CD44-and EpCAM positive CD44-pluripotent stem cell-like cells respectively, further outlining the reprogramming progression. Real-time imaging of the reprogramming cultures after reseeding demonstrates a gradual colony formation corresponding to a logarithmic increase in their confluence and demonstrating the final pluripotent positive and fibroblast negative marker expression at the completion of the reprogramming. Further, using a reporter gene allows the confluence to be measured under phase contrast with a more gradual increase of the GFP confluence observed compared to the total confluence.
Indeed, in this representative experiment, the GFP confluence at day 19 was less than half of the total confluence corresponding to the complete reprogramming. Once mastered, this technique could be used to elucidate information about reprogramming events and track the kinetics of human fibroblast reprogramming. This technique could also be used to look at different starting somatic cell tissues, the reprogramming technologies used, and the medium matrix combinations to elucidate reprogramming events.
After watching this video, you should have a good understanding on how to utilize real-time monitoring and cell surface marker expression, to be able to track the reprogramming of human fibroblasts into iPSCs.
