The protocol demonstrates how to efficiently generate human induced pluripotent stem cells from four milliliters of peripheral blood. On day zero, isolate and expand the mononuclear cells from the peripheral blood sample. Transduce the primary cells with the reprogramming stem co vector.
On day nine, proceed to plate the transduced cells onto inactivated mouse, embryonic, fibroblasts, and culture for about three weeks. Pick the HESC like colonies for expansion and characterization. Results of immunofluorescence staining for the expression of pluripotency markers like SS EEA four, trial one 60 and trial 180 1.
Demonstrate the efficient generation of induced human pluripotent stem cells. The use of the stem cell antiviral vector to generate human induced per potent stem cells offer several advantages over other existing methods. Perhaps most appealing is the overall simplicity of the approach as it requires easy access to donor tissue, and by that I mean readily obtainable foreigners of peripheral blood.
The most important attributes of this method are the efficiency of rep programming and the quality of the obtained IPS cell clones. Demonstrating the procedure will be Andrea January Summer, a postdoctoral fellow in my lab and Sarah Rozelle, a graduate student in George Murphy's lab. All on day zero.
Draw four milliliters of peripheral blood into a vacutainer cell preparation tube with sodium citrate. Invert the tube eight to 10 times within two hours of collection. Centrifuge the sample at 1, 800 G for 30 minutes at room temperature to collect the mononuclear cells.
Aspirate off the plasma and pipette out the buffy coat the cell layer between the gel barrier and plasma. Transfer the Buffy coat to a sterile 15 milliliter conical centrifuge tube and bring the total volume to 10 milliliters with sterile PBS. Invert the tubes several times and centrifuge at 300 G for 15 minutes.
Reus suspend the cell pellet in 10 milliliters of sterile PBS perform a cell count and then transfer one to two times 10 to the sixth cells into a sterile 15 milliliter chronicle centrifuge tube. Harvest the cells by centrifugation and resuspend the pellet in two milliliters of expansion medium. Then seed the isolated mononuclear cells in one well of a 12 Well plate and culture at 37 degrees Celsius and 5%carbon dioxide.
Harvest the remaining cells by centrifugation and freeze stocks of approximately two times 10 to the sixth cells per vial in FBS containing 10%DMSO on days three and six. Transfer the cells to a sterile 15 milliliter conical tube and wash the well once with one milliliter of QBSF 60 stem cell medium. To collect the adherent cells.
Harvest the cells by centrifugation, resuspend the cell pellet in two milliliters of em. Then transfer the cell suspension to one well of a 12 Well plate and return the culture to the incubator on day nine. Transfer the cells to a sterile 15 milliliter conical tube and wash the well once with one milliliter of QB SF 60 stem cell medium to collect the adhere cells centrifuge the culture at 300 G for 10 minutes.
Next, resuspend the cell pellet in one milliliter of fresh EM containing five micrograms per milliliter of poly brain and STEM lentivirus for an MOI of one to 10. Then transfer the suspension to one well of a 12 well plate and centrifuge at 2, 250 RPM for 90 minutes at 25 degrees Celsius. Add an additional one milliliter of fresh EM containing five micrograms per milliliter of poly brain for a total of two milliliters of em and place the plate in the cell culture incubator on day 10.
After harvesting the cells as shown earlier, resus suspend the cell pellet in two milliliters of EM culture. The transduced cells in one well of a 12 well plate on day 11 coat the wells of a six well plate with 0.1%gelatin, then plate inactivated mouse embryonic fibroblasts at two times 10 to the fifth cells per well in meth medium. Prepare three wells per infection.
The next day, harvest the transduced mononuclear cells as shown earlier. After centrifugation resuspend the cell pellet in three milliliters of meth medium containing BFGF. Ascorbic acid and growth factors.
Now aspirate the media from the MES plate one milliliter of the MCs per well OFMs. Then add 1.5 milliliters of complete meth media centrifuge the plate at 500 RPM at 25 degrees Celsius for 30 minutes. Then culture at 37 degrees Celsius and 5%carbon dioxide for two days from day 14 onward.
Replenish the media every other day with 2.5 milliliters of meth media containing 10 nanograms per milliliter of BFGF and 50 micrograms per milliliter of ascorbic acid, but no growth factors. Around day 20, once small colonies appear, feed the cells daily with two milliliters of human embryonic stem cell medium between days 30 and 40. Pick each colony and transfer to individual wells of a 12 Well plate preceded with inactivated maths containing one milliliter per of HESC medium plus rock inhibitor feed cells daily.
Thereafter with one milliliter of HESC medium without the rock inhibitor, the human monocyte derived pluripotent stem cells are now ready for immunofluorescence staining of pluripotency markers and also for excision of the integrated reprogramming cassette. During this protocol, the cells undergo several morphological changes from P BMCs to the generation of human I PSCs. At day zero, the isolated mononuclear cells from peripheral blood are cultured in expansion medium after nine days.
Bunches of grapes like clusters of cells are observed, suggesting that the cells are healthy and proliferating before transduction. Around day 21 week after the cells are plated onto MAs colonies form. At day 30, the IPSC colonies show typical human embryonic stem cell-like morphology.
This immunofluorescence analysis of IPCs generated from pbmc shows the expression of the pluripotency markers SSEA four, trial one 60 and trial 180 1. The I PSCs also stain positive for alkaline phosphatase. These images show the steps to generate transgene free IPCs by transfection with a plasmid coex expressing CRE recombinase, and a pur mycin resistance gene as described in summers etal 2010 compared to IPCs before transfection cell death is observed.
After two days of pur mycin selection, new colonies emerge from resistant cells around 10 days after transfection southern blood analyses on select colonies, confirm the excision of the transgene lanes 1, 3, 5, and seven show the presence of the transgene in IPSC clones before excision. These bams are absent on lanes 2, 4, 6, and eight after excision of the transgene. After watching this video, you should be able to consistently and robustly generate high quality, normal or disease specific human IPS cells from small amounts of peripheral blood from any individual.
This approach simplifies the process of rep programming to make it widely accessible to the stem cell community. At the same time, provides a baseline for the generation of more homogeneous population of stem cell lines.
