In this video article, human adult fibroblasts are reprogrammed to generate pluripotent stem cells using retroviral vectors. First, human fibroblasts are transduced with retroviral vectors encoding the transcription factors. T three four, SOX two, KLF four, and cmic.
The infected fibroblasts are cultured on mouse embryonic feeder cells for 14 to 28 days. The resulting cultures are stained with TRA 180 1 antibody to identify colonies of reprogrammed cells. Positive colonies are then transferred to MEF containing plates for expansion.
Immuno cyto chemical staining confirms the expression of pluripotency markers and establishment of pluripotent stem cell lines. Additional studies should then be performed to further characterize the lines. Generally individuals new to the somatic cell cell programming and human induced pluripotent stem cell cultures will struggle because it is difficult to identify the correct IPSC colonies.
Demonstrating the procedure will be Dr.Sula Pollak post-Doctor O Fellow from my laboratory. Begin this procedure by plating Phoenix AM FO cells at a density of about seven to 8 million cells per 10 centimeter plate in 10 milliliters of high glucose DMEM place in the incubator and culture overnight. The next day prepare transfection mixes consisting of 12 micrograms of a vector encoding either T three four SOX, two klf, four cmic or GFP gene and 35 microliters few gene six in a total volume of 500 microliters of serum free high glucose DMEM with L glutamine.
Incubate the mixtures for 20 minutes. Then to transfect the phoenix cells gently pipette the DNA complexes into the plates containing phoenix cells, which should be 70 to 80%confluent. Incubate the cells at 37 degrees centigrade after six to eight hours.
Aspirate the medium and replace it with eight milliliters of high glucose DMEM with antibiotics. Then return the plates to the incubator for 12 hours following the incubation. Use a pipette to aspirate and collect the virus containing medium.
Then replace the medium and repeat this process every 12 hours until medium has been collected four times. Store the medium at four degrees centigrade. Once all of the medium has been collected, combine all of the portions, then pour it through a 0.45 micrometer tube top filter into a 50 milliliter conical tube to remove detached cells and debris.
The filtered medium, which contains viral particles can be kept in the refrigerator for two weeks without losing infectious activity. Freezing is not recommended. The on the day before the transduction procedure thaw the frozen stock of adult human fibroblasts and add one times 10 to the fifth cells per each well of six well gelatin coated plates.
The next day in a 50 milliliter conical tube, combine 3.5 milliliters each of T four SOX two, klf four and cmic viral medium, and add poly brain to a final concentration of six micrograms per liter as a control. Prepare one milliliter of GFP viral medium, three milliliters of culture, medium and poly brain at a final concentration of six micrograms per milliliter. Aspirate the medium from the plate containing human fibroblasts.
Then add four milliliters of mixed viral media or control medium centrifuge the plates at 1600 G for one hour at 20 degrees centigrade following the centrifugation. Incubate the plates for approximately 12 hours. Then replace the viral medium with fibroblast culture medium and incubate for another 12 hours.
Repeat this process from infection to culture in fibroblast medium, two additional times after the last infection. Culture the fibroblasts in DMEM for 48 hours. Then check the efficiency of the infection of the parallel transductions with the GFP retroviral medium.
Next to initiate reprogramming from somatic to pluripotent cells. The infected fibroblasts are cultured in human embryonic stem cell medium or HES medium seed mouse embryonic, fibroblasts or MEFs at a density of about two times 10 to the fifth cells per well of the gelatin treated. Six well plate in fibroblasts growth medium the following day.
Split the infected human fibroblasts using 0.05%tripsin EDTA and seed them at the density of approximately 1.2 times 10 to the fourth per well in the fibroblast growth medium. The next day. Replace medium with HES media containing 0.5 millimolar sodium butyrate for the initial seven days of the reprogramming.
Change the medium daily. Replace media with HES media without sodium butyrate and continue reprogramming for an additional seven to 21 days. Change the medium daily each day.
Check the transduced cells for morphological changes. Colonies of cells will start to emerge approximately 10 days after transferring the transduced fibroblasts onto feeder cells. The colonies containing the reprogrammed cells are now referred to as human induced pluripotent stem cells or human IPS cells.
The human IPS cell colonies will be ready to be isolated in 14 to 28 days after plating them on the MEF feeder cells. At this stage, human IPS cell colonies can be transferred onto either MEF containing or matrigel covered plates here. Transfer onto the MEF containing plates is demonstrated a day before picking the human IPS cell colonies Seed 24 well plates containing gamma radiated MEF feeder cells in fibroblast growth medium.
Incubate at 37 degrees Celsius with 5%CO2 on the day that the human IPS cell colonies will be picked. Aspirate the fibroblast growth medium from the ME Fs and rinse them with PBS to remove any traces of FBS. Subsequently add 0.5 milliliters of human embryonic stem cell or HES medium containing 10 micromolar rock inhibitor Y 2 7 6 3 2 to each well next, using a microscope mounted in a laminar flow hood, examine the reprogramming plates for the human IPS cell colony formation.
If necessary, remove the fibroblasts surrounding the human IPS cell colonies with a 21 gauge needle. Rinse the plates with PBS and add fresh HES medium containing the TRA 180 1 stain alive specific antibody. Return the cells to the incubator at 37 degrees centigrade with 5%CO2.
After 30 minutes, replace the medium with fresh HES medium supplemented with 10 micromolar rock inhibitor. Then view the colonies under the fluorescent microscope. Mark TRA one to 81 positive colonies.
Using an objective marker using a 21 gauge needle. Cut the TRA 180 1 positive human IPS cell colonies into several small pieces. Next, using an automatic pipette transfer fragments of human IPS cell colonies into individual wells of the 24 well plate with ME Fs.Avoid transferring non IPS cells.
Place the plates in the incubator and allow the human IPS cell colonies to attach for 24 to 36 hours change medium daily. The human IPS cell colonies with correct HE like morphology will be visible 48 hours after the initial transfer onto a 24 well plate manually passage human IPS cell colonies every six to eight days by cutting them with a 21 gauge needle plate. Expanding cultures onto a 12 well and subsequently on a six well plate after clonal expansion.
And establishing the human IPS cell lines analyze expression of pluripotency markers TRA one 60 SSEA four, T three, four SOX two, and nanog using immunochemistry once the lines have been established. Detailed characterization may entail in vitro and in vivo. Differentiation analysis, karyotype analysis, analysis of transgene silencing methylation studies and gene expression studies.
Efficient transduction with retrovirus containing medium is critical for successful reprogramming to determine the efficacy of viral transduction. Human adult fibroblasts derived from a Friedrichs ataxia patient were visualized after two consecutive infections with GFP retroviral medium when the fibroblasts were infected by simple incubation with viral medium, less than 20%of the cells are transduced. However, when addition of the GFP retroviral media was immediately followed by the efficiency of transduction was dramatically increased.
Here more than 80%of the cells are transduced to conduct an initial characterization of the isolated human IPS cell colonies. The expression of pluripotency markers characteristic of human ES cells was assessed by immunochemistry as shown here. Isolated human IPS cell colonies were positive for the pluripotency markers.
T three four SOX two nano SSEA four and TRA one 60 DNA. Cos staining with DPI shown on the upper panels is detected in both the human IPS cell colonies and the feeder cells. These results demonstrate that human IPS cells obtained using this reprogramming protocol express appropriate pluripotency markers characteristic for undifferentiated human pluripotent stem cells.
After watching this video, you should have a good understanding how to successfully reprogram human fibroblast into pluripotent stem cells using retroviral transaction identified correct human induc, pluripotent stem cell colonies, and conduct initial characterization of HI PCs.
