Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

The overall goal of the following experiment is to efficiently generate induced pluripotent stem cells or IPSC from muscular dystrophy patients starting from urine derived cell cultures and an integration free reprogramming method. This is achieved by first collecting urine samples from muscular dystrophy patients. As a second step, the urine cells are isolated and expanded into urine derived cell cultures.

Next, the urine derived cells are transduced with Sendai virus containing Yamanaka factors and typical pluripotent like colonies emerge after 12 days. Ultimately by passage 13, the transgene free state of the pluripotent stem cells can be confirmed by R-T-P-C-R analysis. The main advantage of this technique over existing methods like the reprogramming of isolated skin fibroblasts using lentiviral vectors is that with this technique, the somatic cells can be obtained non-invasively from patients and used to efficiently generate the IPCs using a zero footprint method of viral reprogramming.

This method can help answer key questions in the field of muscular dystrophy, such as how these conditions be modeled in vitro without safety concerns for human application. Demonstrating the procedures will be Mohammad Zishan Al, a postdoctoral fellow Courtney Guesting, a technician, and Melanie Ryder graduate student, all three from my laboratory To collect a urine sample begin by advising the patient to drink fluids 30 minutes prior to the collection to ensure that about 30 to 40 milliliters of urine can be collected. When the patient is ready, give the patient a urine collection kit containing the following items, written instructions on how to obtain sterile or clean catchier and sample moist antibacterial toilets, and a 100 milliliter sterile specimen collection cup.

When the sample has been collected, immediately place the collection cup on ice and transfer the sample to the laboratory in a cooler. Then use sterile pipettes to transfer the urine sample into sterile 50 milliliters centrifuge tubes, and spin the tubes at 400 times G for 10 minutes at room temperature. Next, in a BSL two biological safety cabinet aspirate all but the last one, milliliter of the supernatant, being careful not to disturb the pellet.

Then wash the cells in seven milliliters of washing buffer and spin the cells down again. This time aspirating all but the last about 0.2 to 0.5 milliliters of the supernatant. Now reuse the pellet in two to three milliliters of urinary progenitor cell or UPC medium and transfer the suspension into four to six wells of an uncoated 24 well plate after 72 hours, supplement the culture with 0.5 milliliters of fresh UPC medium per well.

Monitor the culture for between four to six days of incubation. Small two to four cell colonies of rounded or elongated cells representing type one or type two renal epithelial cells respectively should start to appear. Continue to change the medium every two to three days.

As the colonies will undergo a rapid expansion phase at around nine to 15 days of culture, the cells should be 80 to 90%confluent at this point, split the cells onto two to four wells of a 24 well plate for further expansion. Marking this culture as cell passage one, the urine cells can then be characterized for lineage specific markers through flow cytometric analysis, immunohistochemical staining, or through reverse transcriptase polymerase chain reaction to ensure high efficiency reprogramming to days prior to the transduction seed, 60, 000 cells per well. From a passage one to five culture into two wells of a six well plate on day zero.

Prepare the Sendai reprogramming vector by adding each of the four OSKM factors at an appropriate MLI for reprogramming to one milliliter of prewarm UPC media aspirate the suspense media and slowly add the virus suspension to one well of the urine cells for the next six days. Replace the supernatant daily with fresh UPC medium on day five plate mitomycin seed treated MES onto gelatin coated 100 millimeter square culture dishes on day six. Dissociate the urine cells with trypsin, resuspend them in UPC medium and plate them at five times 10 of the fourth to two times 10 of the fifth cells per meth feeder plate.

The next day, replace the UPC medium with human embryonic stage or HES medium and monitor the cells under a microscope to observe the transformed cells around 12 to 18 days. Post transduction, the cells should form clonal aggregates with a characteristic cobblestone morphology and a higher nucleus to cytoplasmic ratio. To identify the reprogrammed IPSC clones, perform live cell staining for a TRA 180 1 antibody.

Then use a 26 gauge half inch needle to crosshatch the TRA 180 1 positive clonal aggregates into four to six small equal sized pieces per clone. Next, use a sterile pipette tip to pick and transfer 10 to 20 cross hatched pieces from multiple clones into each well A 24 well matrigel coated plate with HESC. Medium, 24 to 48 hours after the reprogrammed clones are plated onto the matrigel surface.

Replace the medium with IPSC medium. Use a pipette tip to manually scrape off any differentiated cells or contaminating meth feeder cells to aid in the purification of the fully reprogrammed and pluripotent dystrophic IPSC clones. After the clones have reached about 100 micrometers in size, dissociate the cells with EDTA for three minutes, and then plate small cell aggregates of the reprogrammed dystrophic IPSC, suspended in IPSC medium supplemented with RO kinase inhibitor onto a fresh matrigel coated 12 foil plate.

Finally, confirm the pluripotency of each clone by both gene expression analysis and immunohistochemical staining. For pluripotent markers, most cells isolated from human urine are positive for progenitor and stem cell markers such as CD 44, CD 73, and CD 1 46. The cells also exhibit mesenchymal markers such as alpha smooth muscle actin and menton.

R-T-P-C-R of the isolated cells provides evidence of a mixed population of cells in the cultures. In that there is weak expression of cytokeratin seven and UIN one A and three a markers of euro epithelial cells. The urine cells transform from elongated type two cells on day zero into a cobblestone pattern by day four of reprogramming.

By day 12, typical pluripotent clones are observed and are able to maintain their pluripotent morphology under feeder free conditions. Live cell staining for TRA 180 1 can be used to identify the reprogrammed clones. To confirm the generation of vector and transgene, free pluripotent clones, R-T-P-C-R can be performed using primers against the Sendai virus genome and each of the exogenous OSKM factors.

The exogenous gene expression is no longer detected by passage 13, but the upregulation of endogenous factors can be observed. Immunofluorescent staining confirms the expression of pluripotent markers on the IPCs but not the urine cells. The expression of the dystrophin protein and gene can then be verified by further immunofluorescent staining western blotting and R-T-P-C-R respectively as demonstrated with these representative images from a wild type control and dystrophic cell lines.

Once master, this technique can be employed to achieve ene free reprogramming of ucs. Within a few weeks Following this procedure, we can differentiate the IPCs into cardiomyocytes or skeletal myocytes in order to further our of the mechanisms behind muscular dystrophy.