Developmental Biology
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Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients
Chapters
Summary October 16th, 2016
Here we describe a protocol for generating human induced-pluripotent stem cells from patient-derived fibroblast-like synoviocytes, using a lentiviral system without feeder cells.
Transcript
The overall goal of this experiment is to generate induced-pluripotent stem cells, or IPCs, from fibroblast-like synoviocytes isolated from the synovial tissue of a rheumatoid arthritis inflamed joint. Currently, iPSC's can be generated from various human primary cells. We successfully generated iPSC's from human fibroblast-like synoviocytes, which are the key pathological cells of rheumatoid joints.
This may help to investigate the pathology of rheumatoid arthritis. This periodical can help answer key questions about degeneration of IPSC's, using RA-FLSs. Begin by placing the synovial tissue in a 100 millimeter dish and washing the tissue with 5 milliliters of PBS supplemented with antibiotics.
Remove the yellowish fat tissue and bone residue. Then transfer the trimmed tissue into one well of a six well plate containing 5 milliliters of DMEM supplemented with FBS and use scissors to mince the tissue until the pieces are small enough to penetrate a disposable pipet tip. Transfer the medium to a 50 milliliter conical tube, then add 5 milliliters of fresh DMEM plus FBS to the well to collect any remaining tissue and transfer the entire well contents to the tube.
Next, add ice-thawed collagenase to a final concentration of 0.01%to the tube and seal the tube with parafilm for incubation in a 37 degree Celsius water bath with shaking. After four hours, fill the tube with fresh DMEM plus FBS to a final volume of 50 milliliters and collect the tissue fragments by centrifugation. Remove the supernatant without disturbing the pellet then re-suspend the cells in 40 milliliters of fresh medium for another centrifugation.
Re-suspend the second pellet in 25 miilliters of fresh DMEM plus FBS and allow the large clumps of tissue to settle. Then transfer the supernatant into a new 100 millimeter dish and incubate the synoviocytes at 37 degrees Celsius and 5%CO2 for 14 days. To expand the synoviocyte culture discard the used medium and wash the cells in 5 milliliters of PBS.
Remove the PBS and dissociate the cells with 1 milliliter of EDTA in the cell culture incubator for 2 minutes. At the end of the incubation, tap the dish gently to detach any remaining adherent cells and transfer the resulting cell suspension into a 15 milliliter conical tube for centrifugation. Re-suspend the pellet in 30 milliliters of DMEM and FBS and split the cells into 3 new 100 millimeter dishes.
Feed the synoviocytes with fresh medium every 3 days, splitting the cells into 3 new cultures at 80%confluency as just demonstrated. On transduction Day zero, see 3 x 10 to the 4th FLS's per well onto a 6 well plate in fresh growth medium for an overnight incubation at 37 degrees Celsius in 5%CO2. The next day, thaw 1 vial of Lenti-virus containing 4 Yamanaka factors at 4 degrees Celsius and replace the FLS culture medium with FLS growth medium supplemented with hexadimethrine bromide and ascorbic acid.
When the vial contents have thawed add 30 microliters of lenti-virus to the cells and gently mix to homogeneously distribute the virus. To improve the infection, centrifuge the plate and return the cells to the incubator replacing the medium every day with fresh FLS growth medium containing sodium butyrate and ascorbic acid for 3 days. On the 4th day, replace the medium with a mixture of FLS growth and iPSC medium containing sodium butyrate and ascorbic acid.
On day 5 add 60 microliters of vitronectin to 6 milliliter of PBS without calcium and magnesium and coat 3 wells of a 6 well plate with 2 milliliters of the solution each for at least an hour at room temperature. While the vitronectin is setting, wash the transduced cells with PBS and detach them with EDTA as demonstrated. After collecting the dissociated cells by centrifuguation, re-suspend the pellet in 900 microliters of fresh medium.
Remove the excess vitronectin from the wells and add 300, 150, and 100 microliters of cells into individual wells. Then return the cells to the incubator, replacing the medium daily with fresh iPSC medium until colonies appear. To pick the colonies, first add 500 microliters of iPCS medium, supplemented with ROCK inhibitor to each well of a vitronectin-coated 48 well plate.
Then, place the FLS cultures under a dissecting microscope on a clean bench, and use a 10 microliter pipet tip to cut around a colony of interest. Using a 200 microliter pipet tip, transfer the picked colony to 1 well of the 48 well plate. When all of the colonies have been picked, place the plate in the cell culture incubator until the colonies are big enough for transfer.
Splitting the cells when the colonies are out of the visible field of the microscope, as viewed at 100X magnification. In this image, the morphology of the isolated FLS after an initial 14 day culture can be observed. The cells exhibit the characteristics of fibroblasts in general with a similar expression of the fibrotic markers vimentin and fibronectin as well as a low expression of the macrophage-like synoviocyte marker CD-68.8 to 11 days after the Yamanaka factor lenti-viral transduction, small colonies begin to appear that can be picked and amplified for 5 to 10 passages.
These rheumatoid arthritis iPSC's express alkaline phosphatase indicating that they remain undifferentiated. The cells also express pluripotent markers as confirmed by RT-PCR and immunofluorescence analysis. Rheumatoid arthritis iPCS's exhibit a normal chromosomal pattern of 44+XY as determined by karyotyping.
Furthur, 12 weeks after their injection into SCID mice, the rheumatoid arthritis iPSC formed teratoma, that display diverse tissue phenotypes. With their germ layer differentiation confirmed by immunofluorescent staining. Once mastered, iPSC's can be generated from FLSs with synovium.
After watching the video, you should have a better understanding of how to isolate from synovial tissue and how to induce reprogramming using them. These RAF-FLS-derived iPSC's can be useful for studying regeneration or drug screening in rheumatology. Once these iPSC's are established, various kinds of target cells or organs can be generated.
Which may be useful for studying the disease or other future applications, such as gene editing and so forth.
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