Reprogramming Induced Pluripotent Stem Cell Lines from Frozen Buffy Coat Samples

The purpose of this protocol is to reprogram human frozen buffy coats or PBMCs into induced pluripotent stem cells. This protocol uniquely purifies buffy coats after thawing and identifies a somatic cell morphology that is indicative of reprogramming success. To begin, retrieve the frozen buffy coat derived from human blood and place it on ice.

Gently swirl the sample in a 37 degrees Celsius water bath and remove the vial when only a small ice crystal remains. Carefully transfer the contents of the vial dropwise into a conical tube containing 12 milliliters of DPBS. Rinse the vial with one milliliter of DPBS, and add the rinse to the conical tube containing the retrieved cells.

Centrifuge the sample using a swinging-bucket rotor at 200 g at room temperature for 10 minutes. Carefully remove the supernatant, and resuspend the cell pellet in four milliliters of DPBS containing 1%FBS. Next, gently layer the buffy coat mixture over three milliliters of density gradient solution, avoiding mixing at the interface.

Centrifuge using a swinging-bucket rotor at 800 g for 20 minutes at room temperature with the brake off. Using a pipette, remove and discard the upper plasma layer without disturbing the plasma to gradient interface. Then remove the peripheral blood mononuclear cell, or PBMC layer at the interface and transfer it to a clean 15-milliliter tube.

Wash the PBMCs by adding three milliliters of expansion medium and centrifuging at 200 g for 10 minutes at room temperature. Carefully remove the supernatant and resuspend the pellet in two milliliters of expansion medium containing cytokines such as SCF, IL-3, EPO, and IGF-1. Plate the cells in 1 well of a 12-well plate.

Culture the cells at 37 degrees Celsius with 5%carbon dioxide and 20%oxygen for 10 to 14 days. Replace half of the medium every other day until a dense population of bright, round erythroblasts is observed, indicating that the cells are ready for transduction. Three days after transduction with Sendai virus carrying the Yamanaka factors, plate the cells onto mouse embryonic fibroblasts at two milliliters per well.

Incubate the cultures for about seven days, feeding emerging colonies with hESC media every other day. Place the plate containing the transduced cells and emerged colonies under a dissecting microscope. Use the microscope to locate one colony to pick and lift the lid of the plate to expose the well.

Aseptically open the syringe and expose the needle. Guided by the microscope, use the needle tip to detach the colony from surrounding feeder cells by tracing the outline of the colony. Use the needle tip to cut the colony into a few equal-size pieces.

If the pieces have not detached, use the side of the needle to scoop or scrape them off the plate until they are floating. Now using a P200 pipette, locate the picked colony under the microscope, aspirate the floating colony with the pipette, and transfer it to a well of the prepared 24-well plate. Ensure the entire colony is transferred without collecting other colonies or feeder cells.

Place the plate in the incubator at 37 degrees Celsius with 5%carbon dioxide and 20%oxygen until the desired confluency is achieved. Identify colonies that are large, round and exhibit glowing edges as ready to split. Aspirate the media from the well and rinse the sample once with one milliliter of DPBS.

Then remove the DPBS and add 500 microliters of 0.5 millimolar EDTA dissociation buffer. Incubate the sample at 37 degrees Celsius with 5%carbon dioxide for two minutes until colonies loosen and cells round up. Next, remove the plate from the incubator and aspirate the EDTA dissociation buffer.

Using a P1000 pipette, collect the cells with one milliliter of human pluripotent stem cell medium by gently pipetting up and down until colonies are detached and slightly broken up. Transfer the one milliliter of medium and cells into a new six-well plate containing one milliliter of fresh human pluripotent stem cell medium. Place the cells in the incubator at 37 degrees Celsius with 5%carbon dioxide and 20%oxygen.

Feed induced pluripotent stem cells daily until the well is confluent and ready to split. Meanwhile, coat a 10-centimeter dish with 7 milliliters of five micrograms per milliliter vitronectin solution prepared in DPBS and incubate it at room temperature for one hour. To continue splitting, add 7 to 10 milliliters of human pluripotent stem cell medium to the 10-centimeter dish and transfer cells from the six-well plate to the dish at a 1:1 ratio, and incubate as demonstrated earlier.

Confirm that the cells in the 10-centimeter dish are confluent before splitting the cells again. Adjust the splitting ratio based on cell morphology and density, typically using a 1:10 ratio, and note the passage number at each passage. Finally, characterize induced pluripotent stem cells by confirming stemness marker NANOG expression using immunofluorescence.

Differentiate the cells into ectoderm, mesoderm, and endoderm lineages using defined culture conditions. Assess differentiation potential by staining for PAX6, Brachyury, and SOX17, along with loss of NANOG expression. At the end of expansion, PBMCs differentiated into erythroblasts appearing as bright, round cells of large and small size.

Whereas cultures lacking these cells or appearing dead were unlikely to successfully reprogram. Successful transduction and reprogramming were marked by the emergence of induced pluripotent stem cell colonies on irradiated feeder cells, which then developed typical stem cell morphology. Whereas cells that did not adopt stem cell-like morphology were not successfully reprogrammed.

Successfully reprogrammed induced pluripotent stem cells exhibited dense, round colonies with smooth, bright edges. Subclones with less ideal morphology, such as spontaneous differentiation or dense centers, were cleaned up by adjusting splitting conditions. Some picked colonies did not successfully expand.

In addition to morphology, induced pluripotent stem cells exhibited stemness as evidenced by expression of stemness marker NANOG. Reprogrammed induced pluripotent stem cells demonstrated differentiation potential into ectoderm, mesoderm, and endoderm. This protocol provides detailed checkpoints to track and predict reprogramming success.

To further confirm that an iPSC line is suitable for use, karyotyping, genotyping, and regular mycoplasma testing should be performed. This protocol allows for reprogramming from existing patient populations with biobanked buffy coats, broadening the utility of iPSC technology.