Reprogramming Induced Pluripotent Stem Cell Lines from Frozen Buffy Coat Samples

Summary

The current protocol details steps for troubleshooting induced pluripotent stem cell (iPSC) derivation from patient samples’ peripheral blood mononuclear cells (PBMCs) that have been difficult to reprogram following the manufacturer’s instructions. This protocol is specific to frozen buffy coat samples but may also be applied to the reprogramming of purified PBMCs.

Abstract

Human pluripotent stem cells (hPSCs) are a valuable tool for disease modeling. Further stem cells can be reprogrammed from adult somatic cells, called induced pluripotent stem cells (iPSC). iPSC technology allows for the evaluation of specific study participants and populations and ventures into personalized medicine. Blood is routinely taken and cryopreserved for research purposes. These samples are processed either as buffy coats, a blood sample containing white blood cells and platelets, or as peripheral blood mononuclear cells (PBMCs), which represent a more purified population of white blood cells without eosinophils, basophils, platelets, or red blood cells. Both are a readily available and relatively non-invasive source for reprogrammable somatic cells. Several reports detail reprogramming from PBMCs, whereas only one describes this process from frozen buffy coats. Recent experience revealed PBMC reprogramming protocols available in the literature and from manufacturers to be unsuccessful when applied to frozen buffy coat samples, necessitating the adaptations and troubleshooting strategies described here. For many researchers, who employ iPSC technologies, it is imperative to have thorough protocols with a high success rate, especially in cases, where patient samples may contain only few cells, are obtained in wide time intervals, are from limited participant pool, or are otherwise highly valuable. Here, checkpoints and troubleshooting strategies are identified to increase the chance of reprogramming human frozen buffy coats or purified PBMCs. Ultimately, this protocol will allow researchers to identify predictors of reprogramming success and strategize alternative approaches to improve the chances of successful iPSC derivation.

Introduction

Human pluripotent stem cell (hPSC) technology is an easily scalable and renewable source of human cells that can be differentiated into any cell type in the body, allowing researchers to investigate cell and tissue types that are hard to obtain or inaccessible in patients. Further, induced pluripotent stem cells (iPSCs) can be derived directly from patient somatic cells¹. This allows for the differentiation of patient-specific cell types of interest, an invaluable tool for disease modeling and drug discovery. Thus, rigorous, reproducible and successful methods to reprogram somatic cells into iPSCs are critical.

Fibroblasts, urine cells, and blood cells^2,3, each with their own limitations, are some of the most frequently used somatic cell sources for iPSC derivation. Fibroblasts, historically the most popular choice for reprogramming³, are not very easily obtained, requiring a somewhat invasive skin biopsy. Reprogrammable cells can be derived from urine samples, which are easy to obtain and non-invasive, but the reprogramming process must begin with fresh samples up to 48 h after collection⁴. Blood offers a great source for reprogrammable cells, as it is minimally invasive to obtain and readily available. There are many available reprogramming protocols from peripheral blood mononuclear cells (PBMCs), blood cells with a single nucleus including lymphocytes, monocytes and natural killer cells⁵. Because PBMCs are less dense than other blood cells, they can be isolated/purified from freshly isolated whole blood samples through density gradient centrifugation⁵. Alternatively, upon centrifugation, whole blood separates into three distinct layers: plasma, buffy coat, and red blood cells⁶. Buffy coats contain PBMCs as well as platelets and some erythrocytes. However, PBMCs are less readily available in most biobanks, whereas whole blood or buffy coats are abundant and accessible⁷. Despite the availability of buffy coats in biobanks and the presence of reprogrammable PBMCs in these samples, very few protocols detail reprogramming from frozen buffy coats⁸, leaving this an untapped resource.

Currently, there are many published protocols^1,8,9 and commercially available kits¹⁰ to streamline reprogramming; however, they do not deeply troubleshoot how to maximize success for patient donor cells that are hard to reprogram. It is commonly accepted that some cells just won’t reprogram, and when this happens, many researchers may try with a different donor cell type or a different patient. However, this may not be a viable option for many researchers or patient populations. Unlike previously published protocols^8,9, this protocol includes a guide with check points and criteria, based on which to predict reprogramming success and takes a unique approach by purifying frozen buffy coats with density gradient centrifugation upon thawing.

The process begins with drawing blood from chosen individuals and isolating either PBMCs or buffy coats. For new experiments, this is likely to include recruiting new donors who will undergo fresh blood draws under IRB approval and with a proper consent form. The next step is somatic cell expansion, in which frozen blood cells are thawed and cultured under conditions that promote the proliferation of hematopoietic stem cells and erythroid progenitors, the blood cells that are the best candidates for reprogramming¹¹. After 10–14 days, cells should be ready for reprogramming, as evidenced by a distinct morphology which is not discussed in previous protocols, and are then transduced by inoculation with a Sendai virus carrying the Yamanaka factors Oct4, Sox2, c-Myc, and Klf4¹. Following transduction, cells will begin expressing the Yamanaka transcription factors and will return to a pluripotent state, again denoted by a distinct morphological change and proliferation into stem cell colonies. Emerging colonies are then individually picked, expanded, and cryopreserved. After ten passages, characterization by confirmation of expression of stemness markers and differentiation potential into the three germ layers validates a successfully reprogrammed iPSC line.

This protocol works best for cryopreserved buffy coats, which have been stored for < 1 month, or cryopreserved PBMCs stored for up to 2 years with at least 1 million viable cells upon thawing. However, the described checkpoints may help guide troubleshooting reprogramming from any quality blood sample. Additionally, details about adapting reprogrammed iPSCs to feeder-free conditions can be applicable to any iPSC reprogramming workflow, regardless of starting somatic cell type.

This protocol, established through hands-on troubleshooting, details reprogramming iPSCs from frozen buffy coats and identifies morphological indicators of reprogramming success, providing a workable guide to reprogramming difficult samples. Ultimately, implementing these strategies results in conservation of time, money, reagents, and samples.

Protocol

This protocol follows the guidelines of the University of Georgia’s human research ethics committee. All human subject studies were performed according to the guidelines of the World Medical Association’s Declaration of Helsinki. Based on self-report, the volunteers did not suffer from the following diseases: cystic fibrosis, chronic obstructive pulmonary disease, tuberculosis, asthma, diabetes or acute airway infections. Volunteers of ages between 18–50 years and of both sexes were recruited. Pregnant women were excluded from the study.

1. Buffy coat or PBMC extraction and cryopreservation

Decision criteria: If the samples will be stored for a long time, it is best to choose option 2. If the reagents for PBMC purification are not available, or samples will be stored for a short period of time, option 1 is suitable.

1. Option 1: Buffy Coat Extraction
   1. Perform a phlebotomy (vein puncture to take blood) according to WHO best practices¹² and take 7–10 mL whole blood into anti-coagulant coated blood collection tubes. Keep samples on ice.
   2. Transfer 7–10 mL of whole blood to a 15 mL conical tube. Spin blood down using a swinging bucket centrifuge at 200 x g for 15 min at 4 °C with the brakes off. This should form 3 layers with yellow plasma on top, the white buffy coat layer in the middle, and the red erythrocytes at the bottom.
   3. Collect the plasma using a sterile serological pipette without disturbing the buffy coat layer. Discard the plasma. Transfer the white buffy coat layer to a fresh 15 mL conical tube (approximately 1–2 mL).
      CAUTION: Dispose of the plasma in the appropriate biohazardous waste bin.
      NOTE: Some plasma contamination of the buffy coat is fine as it aids in recovering after thawing.
   4. Add DMSO to the buffy coat such that the final composition is 10% DMSO v/v and freeze in cryovial (1 mL/vial) at -80 °C immediately. The day after, move the vials to liquid nitrogen for long-term storage.
      NOTE: As an example, for 2 ml of buffy coat, add 200 µl of DMSO, mix well, then aliquot into 2 vials. This typically results in 1–3 frozen vials per sample.
2. Option 2: PBMC Purification
   1. Perform a phlebotomy (vein puncture to take blood) according to WHO best practices¹² and take 7–10 mL whole blood into anti-coagulant-coated blood collection tubes. Keep samples on ice.
   2. Add 4 mL density gradient solution 1 into a fresh 15 mL conical tube. Carefully layer 4 mL of blood on top of the density gradient solution 1.
   3. Centrifuge using a swinging bucket centrifuge at 400 x g at RT for 30 min, turning the brakes off. This will result in plasma in the top layer, PBMCs in the middle layer, and the remainder of the blood will be in the bottom.
   4. Collect the PBMC fraction into a fresh 15 mL conical tube, then wash it 2–3 times with 1x PBS by centrifuging in a swinging bucket centrifuge at 350 x g at RT for 5 min with the brake on. Discard the supernatant. To skip cryopreservation and immediately begin reprogramming, proceed to step 2.3.4.
      CAUTION: Dispose of the supernatant in the appropriate biohazardous waste bin.
   5. Resuspend pellet in cryopreservation media (90% serum and 10% DMSO) and freeze in cryovial (1 mL/vial) at -80 °C immediately. The day after, move the vials to liquid nitrogen for long-term storage.
      NOTE: PBMC isolating blood collection tubes may be used rather than density gradient centrifugation for PBMC purification from fresh blood. It is also possible to cryopreserve using commercially available alternate freezing mediums. Typical yield is 1–4 frozen vials per sample.

2. Prepare samples for somatic cell expansion

1. Media preparation
   1. Prepare Expansion Media (EM)⁹ by aliquoting 45 mL of EM base. Add 127.8 µL L-ascorbic acid (50 µg/mL from 100 mM stock) and 450 µL penicillin-streptomycin (100x stock).
   2. Prepare EM + cytokines¹³ to 4 ml EM, add 2 µL stem cell factor (SCF) (50 ng/mL from 100 µg/ml stock), 2 µL interleukin-3 (IL-3) (10 ng/mL from 20 µg/mL stock), 16 µL erythropoietin (EPO) (2 U/mL from 500 U/mL stock), 4 µL insulin-like growth factor 1 (IGF1) (40 ng/mL from 40 µg/ml stock), and 4 µL dexamethasone (1 µM from 1 mM stock). Use EM + cytokines on the same day it is prepared.
      NOTE: Alternative commercial expansion medium systems may be used.
2. Option 1: PBMC Purification from Frozen Buffy Coat
   Decision criteria: if option 1 in step 1 was chosen (i.e. buffy coat extraction), then choose option 1 in step 2 (i.e. PBMC purification from frozen buffy coat).
   1. Retrieve density gradient solution 2 and allow to come to room temperature. Mix thoroughly by inverting the bottle. Aseptically transfer 3 mL density gradient solution 2 to a 15 mL conical tube.
   2. Prepare another 15 mL conical tube with 12 mL Dulbecco’s phosphate buffer saline (DPBS).
   3. Retrieve the vial of frozen buffy coat from the liquid nitrogen. Keep on ice until ready to thaw. Gently thaw the sample by swirling in 37 °C water bath. Remove when only a small ice crystal remains.
   4. Carefully (dropwise) transfer the contents of the vial into the conical tube containing DPBS. Rinse the vial with 1 mL of DPBS and add to the conical tube containing retrieved cells.
   5. Centrifuge using a swinging bucket centrifuge at 200 x g at RT for 10 min. Carefully remove supernatant and resuspend cell pellet in 4 mL DPBS with 1% fetal bovine serum (FBS).
   6. Gently layer the buffy coat mixture on top of the density gradient solution, being careful not to allow mixing. Centrifuge in a swinging bucket centrifuge at 800 x g for 20 min at RT with the brake off.
   7. With a pipet, remove and discard the upper plasma layer without disturbing the plasma:gradient interface. With a pipet, remove the PBMC layer at the interface (Figure 1A) and transfer to a clean 15 mL tube.
      CAUTION: Dispose of the other layers in the appropriate biohazardous waste bin.
   8. Wash the PBMCs with 3 mL EM by centrifuging at 200 x g for 10 min at RT. Carefully remove the supernatant and resuspend the pellet in 2 mL EM + cytokines. Plate in 1 well of a 12-well plate.
   9. Place the plate in the incubator at 37 °C, 5% CO₂ and 20% O₂.
3. Option 2: Thawing frozen PBMCs
   Decision criteria: if option 2 in step 1 was chosen (i.e., PBMC purification), or if there is a concern that frozen buffy coats will have low viability (i.e., in storage for more than 1 month) (Figure 1B), then choose option 2 in step 2 (i.e., thawing frozen PBMCs).
   1. Prepare a 15 mL conical tube with 12 mL DPBS.
   2. Retrieve the vial of frozen PBMCs from liquid nitrogen. Keep on ice until it is ready to thaw. Gently thaw the sample by swirling in a 37 °C water bath. Remove when only a small crystal is left.
   3. Carefully (dropwise) transfer the contents of the vial into the conical tube. Be sure to retrieve all cells from the vial by rinsing the vial with 1 mL of DPBS.
   4. Centrifuge in a swinging bucket centrifuge at 200 x g at RT for 10 min. Carefully remove the supernatant and resuspend the pellet (Figure 1C) in 2 mL EM, then take 10 µL to perform trypan blue exclusion test for cell viability to obtain cell count¹⁴.
      CAUTION: Dispose of the supernatant in the appropriate biohazardous waste bin.
   5. Plate 1.0 x 10⁶–2.0 x 10⁶ cells/mL in 2 mL EM + cytokines in one well of a 12-well plate. Place the plate in the incubator at 37 °C, 5% CO₂ and 20% O₂.

3. Somatic cell expansion (1 day after thawing)

1. Carefully remove 1 mL of spent media from each well. Spent media can be collected in another well or centrifuge tube and checked to ensure that no cells have been lost.
2. Replace spent media with 1 mL of fresh EM + cytokines.
3. Repeat steps 3.1 and 3.2 and take pictures of PBMCs every other day until a significant change in morphology is observed, with bright and round cells of large and small size (Figure 2A, orange and blue arrow, respectively), which are the erythroblasts, the cell type to reprogram. This will take about 10–14 days.
   NOTE: Cells that do not display these morphologies, typically lacking the larger round cells (Figure 2B), are unlikely to reprogram successfully and may be dead (Figure 2C).

4. Transduction (10–14 days after thawing)

1. Prepare Sendai Virus
   1. Using the lot-specific titer value, calculate the volume of each virus needed for transduction of 2.0 × 10⁵ cells using KOS: c-Myc: Klf4 with a multiplicity of infection (MOI) of 5:5:3, as recommended by the manufacturer. The equation for this calculation can be found in the product documents provided by the manufacturer.
   2. Retrieve viruses from the freezer, keeping them on ice until time to thaw. One by one, gently thaw the viruses by swirling in a 37 °C water bath.
   3. Prepare aliquots in which all three viruses are mixed at the appropriate volume for transduction of 2.0 × 10⁵ cells at MOIs of 5:5:3. Freeze aliquots at -80 °C until ready for use.
      CAUTION: Be sure to thoroughly bleach all waste that has contacted Sendai Virus.
      ​NOTE: Manufacturer instructions recommend an actual count of cells with MOI calculation to determine volume of virus added, but the approximation of 2.0 × 10⁵ has been successful for the purposes of this protocol. Aliquoting can be more cost effective and reduces the amount of freeze/thaw cycles for viruses.
2. Day of Transduction (d0)
   1. If the PBMCs were not fed the day before, remove 1 mL of spent media and replace with 1 mL of fresh EM + cytokines. Thaw the virus aliquot on ice, then add directly to the well, swirl the media.
   2. Wrap the plate in parafilm and centrifuge the plate in a swinging bucket centrifuge at 350 x g at RT for 90 min. Place the plate in the incubator at 37 °C, 5% CO_2, and 20% O₂ overnight.
      ​CAUTION: Be sure to thoroughly bleach all waste that has contacted Sendai Virus.
3. 1 Day after transduction (d1)
   1. Collect the cells into a 15 mL conical tube. Rinse well with 2 mL EM and check under the microscope to ensure that all cells have been collected. Centrifuge cells at 300 x g at RT for 10 min.
   2. Carefully remove supernatant and resuspend in 2 mL EM + cytokines. Place in a fresh well of a 12-well plate and return the plate to the incubator at 37 °C, 5% CO_2, and 20% O₂.
      CAUTION: Be sure to thoroughly bleach all waste that has come in contact with Sendai Virus.

5. Replate Transduced cells onto Feeder Cells

1. Prepare Feeder Cell
   1. To 43.5 mL Dulbecco’s modified Eagle medium (DMEM) add 5 mL FBS (final concentration 10%), 0.5 mL minimum essential medium non-essential amino acids solution (MEM-NEAA) (from 100x stock), 50 µL 2-mercaptoethanol (55 µM from 55 mM stock), and 0.5 mL antibiotic-antimycotic (from 100x stock).
   2. Prepare gelatin-coated 6-well plates by adding 600 µL gelatin (0.1% from 2% stock) to 12 mL DPBS and distributing 2 mL per well. Incubate at room temperature for 20 min.
   3. Retrieve frozen vial of irradiated mouse embryonic fibroblasts (MEFs) and gently thaw by swirling in 37 °C water bath.
   4. Add dropwise to 15 mL conical tube containing 10 mL DPBS and rinse vial with an additional 1 mL DPBS. Centrifuge at 200 x g at RT for 5 min.
   5. Carefully remove supernatant and resuspend the cell pellet in 1 mL fibroblast media. Remove 10 µL to perform trypan blue exclusion test for cell viability to obtain the cell count¹⁴.
   6. Remove the gelatin solution from the 6-well plates and plate irradiated MEFs at 2.5 × 10⁴ cells/cm². Incubate in incubator at 37 °C, 5% CO₂ and 20% O₂ for 6–48 h.
      NOTE: Feeder cell plates must be prepared prior to replating of transduced cells. Best results have been seen when irradiated MEFs are incubated for about 2 days before replating, but incubation for at least 6 h can be sufficient.
2. Re-plate Transduced Cells
   1. Prepare iPSC media 90 mL DMEM/F12 with 10 mL FBS (10% final concentration) , 1 mL MEM-NEAA (from 100x stock), 1 mL L-glutamine (2 mM from 200 mM stock), 1 mL penicillin-streptomycin (from 100x stock), 100 µL 2-mercaptoethanol (55 µM from 55 mM stock), and 100 µL fibroblast growth factor 2 (FGF2) (10 ng/mL from 10 µg/mL stock). Add L-ascorbic acid (50 µg/mL) fresh at each feeding.
   2. On day 3 after transduction, collect all media and cells from the transduced well into a 15 mL conical tube. Rinse well with at least 1 additional mL of EM and centrifuge in a swinging bucket centrifuge at 300 x g at RT for 10 min.
   3. Resuspend the pellet in 6 mL iPSC media. Aspirate the media from 3 wells of the plate with irradiated MEFs.
   4. Plate transduced cells onto these 3 wells of irradiated MEF (2ml/well). Incubate in incubator at 37 °C, 5% CO₂ and 20% O₂.

6. Maintenance until colony emergence

1. On days 4, 5, and 6 after transduction, add 1 mL of fresh iPSC media to each well each day. This limits unnecessary disturbance of the freshly reprogrammed cells as they settle onto the feeders, while also providing them with fresh nutrients.
2. On day 7 after transduction, remove all old media and replace with 2 mL of fresh iPSC media per well. Continue to feed with iPSC media every other day until colonies begin to emerge.
3. Prepare hES media 78 mL DMEM/F12 with 20 mL FBS free supplement (20%), 1 mL MEM-NEAA (100x), 1 mL L-glutamine (2 mM from 200 mM stock), 1 mL penicillin-streptomycin (100x), 100 µL 2-mercaptoethanol (55 µM from 55 mM stock), and 100 µL FGF2 (10 ng/mL from 10 µg/ml stock). Add L-ascorbic acid (50 µg/mL) fresh at each feeding.
4. Once small colonies have begun to emerge, continue maintaining the transduced cells by feeding every other day with hES media (Figure 3A).
   NOTE: Colonies will begin to appear as early as 7 days after transduction. If no colonies have emerged by three weeks after transduction, it is not likely that colonies will ever emerge.

7. Pick colonies once they have grown to a larger size (Figure 3B)

NOTE: Sometimes transduced cells undergo a morphological change that does not result in a stem cell like colony (Figure 3C). This is not considered a colony that could lead to a successful iPSC line.

NOTE: From colony emergence, iPSC lines will need to be adapted to the preferred hPSC culturing conditions of the lab. This protocol will demonstrate how iPSC lines have been adapted to feeder-free conditions, cultured on vitronectin (VTN) coated plates, fed with chemically defined and commercially available hPSC media. If colonies are not adapting well to the new conditions, they may need a gentler transition, like being picked onto feeder cells with hES media.

1. Prepare VTN-coated plates by thawing and thoroughly mixing VTN. For a 24-well plate, prepare 12 mL of DPBS with 120 µL VTN (5 µg/mL from 500 µg/mL stock) and distribute 0.5 mL VTN solution/well. Incubate at room temperature for 1 h.
2. Prepare hPSC medium by thawing 1 bottle of hPSC supplement at 4°C overnight. Once thawed, add 10 mL hPSC supplement to one 500 mL bottle of hPSC basal medium.
3. Prepare the plates for picking by removing VTN solution from wells of the 24-well plate and adding 0.5 mL hPSC media with 0.5 µL Y-27632 (10 µM from 10 mM stock) to each well.
4. Bring the new plate, the 6-well containing the transduced cells/colonies, sterile syringe needles, and a p200 pipette and tips to a dissecting microscope in sterile conditions.
5. Place the plate containing the transduced cells and emerged colonies on the dissecting microscope and use the microscope to locate one colony to pick. Lift the lid of the plate to expose the well.
6. Aseptically open the syringe and expose the needle. Guided by the microscope, use the needle tip to detach the colony from any surrounding feeder cells by tracing the outline of the colony with the needle.
7. Use the needle tip to cut the colony into a few equal size pieces. If the colony pieces have not already detached from the plate, use the side of the needle to scoop or scrape them off the plate so they are floating.
   CAUTION: Discard the needle in the appropriate sharps container.
8. With the p200, use the microscope to relocate the picked colony. Aspirate the floating colony with the pipette and transfer to a well of the prepared 24-well plate. Try to get the entire colony into the well without getting any other colonies or feeder cells.
9. Place the plate in the incubator at 37 °C, 5% CO₂ and 20% O₂.
10. Feed the picked colonies with 1 mL of hPSC medium every day until they are confluent.
    NOTE: Colonies have the potential to be genetically distinct from one another. Each well of the 24-well plate must only contain one picked colony. Each picked colony is given a distinct identifier and from here on out is treated as a different cell line from other picked colonies. For a rigorous round of reprogramming, pick at least 24 colonies and expand and characterize at least 3.

8. Expand

1. hPSC maintenance
   1. The picked colonies are ready to split when they are large and round with glowing edges (Figure 4A). Colonies break into multiple pieces when picked. If there are multiple colonies in one well, split them before they have grown large enough to contact each other and begin to differentiate.
   2. Prepare a VTN-coated 6-well plate: For a 6-well plate, prepare 12 mL of DPBS with 120 µL VTN (5 µg/mL from 500 µg/mL stock) and distribute 1 mL VTN solution/well. Incubate at room temperature for 1 h. VTN solution can be replaced with DPBS and stored at 37 °C, 5% CO₂ for a month. In the wells that will be split into, aspirate DPBS and replace with 1 mL hPSC medium.
   3. Aspirate the media from the well to split, then rinse once with 1 mL DPBS. Aspirate the DPBS and add 500 µL 0.5 mM EDTA dissociation buffer (prepared in 500 mL DPBS with 0.9 g NaCl and filter sterilized)¹⁵. Incubate at 37 °C, 5% CO₂ for 2 min, until the colonies start loosening up, and cells inside each colony start to round up and detach from one another.
   4. Remove the plate from the incubator, aspirate the EDTA dissociation buffer. Using a p1000 pipette, collect the cells with 1 mL hPSC medium by gently pipetting up and down until the colonies are fully detached and slightly broken up. Transfer the 1 mL of medium and cells into the new 6-well containing 1 mL of fresh hPSC medium. This first split is done at a 1:1 splitting ratio, all cells from the original plate are carried over to the new plate.
   5. Place the cells in the incubator at 37 °C, 5% CO₂ and 20% O₂. Feed iPSCs daily until the well is confluent and ready to split.
      NOTE: Poor stem cell morphology (Figure 4B) may be mitigated by adjusting splitting protocol such as splitting earlier, using a shorter EDTA incubation time, or adjusting the splitting ratio.
   6. Expand into 10 cm dish.
      1. Once cells are confluent in each well of the 6-well plates, coat a 10 cm dish with VTN (7 mL DPBS with 70 µL VTN (5 µg/mL from 500 µg/mL stock), incubated for 1 h at RT).
      2. Repeat splitting protocol, this time expanding from the 6-well into the 10 cm tissue culture dish (splitting ratio 1:1). Use 7–10 mL of hPSC medium per 10 cm dish.
      3. Once cells in the 10 cm dish are confluent, repeat this splitting process (passaging). At each passage, note the passage number. Splitting ratio depends on cell morphology and density. A typical ratio is 1:10, where one tenth of the cells from the original plate are carried over to the new plate.
2. Freezebacks and stock preparations
   1. After about 3 passages, expand into four 10 cm tissue culture dishes for early passage stocks. Once cells in the 10 cm dishes are confluent, they are ready to be frozen.
   2. Prepare cryovials and place them in the freezer to cool. Be sure the label includes the cell line, clone, passage number, and date of freeze.
   3. Prepare freezing media: 90% hPSC medium and 10% DMSO, you’ll need 1 mL/vial (3 frozen vials per 10 cm dish).
   4. Perform splitting protocol as normal, using 4 mL 0.5 mM EDTA dissociation buffer for each 10 cm dish.
   5. After EDTA incubation, aspirate EDTA. Gently add 10 mL of hPSC medium to the dish.
   6. Gently wash the cells off the dish by pipetting the media. The cells will come off the floor of the dish easily and mix in the media. Only pipette 2–5 times to gently break up colonies but avoid dissociation into single cells.
   7. Centrifuge for 5 min at 200 x g at RT. Aspirate supernatant and resuspend the cell pellet in freezing medium.
   8. Insulate and freeze immediately at -80 °C. Transfer the frozen vials to the liquid nitrogen tank for long-term storage.
   9. Freeze more stocks in the same way once the characterization has been completed.

9. Characterization of iPSCs:

1. Expression of stemness markers. After expanding for at least 10 passages, when colonies in a 24-well are ready to be split, fix with 4% paraformaldehyde and stain by immunohistochemistry for stemness marker NANOG. It will take at least 10 passages for colonies to be Sendai Virus (SeV) negative, which can be confirmed by immunofluorescence (Figure 4C).
2. Test pluripotency ability: Three Germ Layer Differentiations. iPSC pluripotency is confirmed by successful differentiation into each of the three germ layers. Each differentiation starts from a confluent 10 cm dish exhibiting good hPSC morphology (Figure 4A).
   1. Ectoderm¹⁶
      1. The day before the differentiation starts (day-1): Seed iPSCs on matrix-coated 24-well plates at a density of 2.6 × 10⁵ cells/cm² in hPSC medium with Y-27632 (10 µM final concentration) and XAV-939 (2 µM final concentration).
      2. Day 0–day 1: Feed with 1 mL/well differentiation base with LDN (100 nM final concentration), SB (10 µM final concentration), and XAV (2 µM final concentration).
      3. Day 2–day 7: Feed with 1 mL/well differentiation base with LDN (100 nM final concentration) and SB (10 µM final concentration). From d3, feed every other day.
      4. Day 8: fix with 4% paraformaldehyde and stain by immunohistochemistry for ectoderm marker PAX6¹⁷ and confirm loss of pluripotency marker NANOG (Figure 4D).
   2. Mesoderm¹⁸:
      1. The day before the differentiation starts (day -1): Seed iPSCs on matrix-coated 24-well plates at a density of 2.5 × 10⁵ cells/cm² in hPSC medium with Y-27632 (10 µM final concentration).
      2. Day 0: Feed with DMEM/F12 with L-Ascorbic Acid (64 µg/mL final concentration), Sodium Selenite (13.6 ng/mL final concentration), Holotransferrin (10 µg/mL final concentration), chemically defined lipid concentrate (100x), and CHIR (5 µM final concentration).
      3. Day 1: Feed with DMEM/F12 with L-Ascorbic Acid (64 µg/mL final concentration), Sodium Selenite (13.6 ng/mL final concentration), Holotransferrin (10 µg/mL final concentration), chemically defined lipid concentrate (100x), and heparin (0.6 U/mL final concentration).
      4. Day 2: Fix with 4% paraformaldehyde and stain by immunohistochemistry for BRACHYURY-T¹⁹ and confirm loss of pluripotency marker NANOG (Figure 4E).
   3. Endoderm²⁰​
      1. The day before the differentiation starts (day -1): Seed iPSCs on matrix-coated 24-well plates at a density of 1.0 × 10⁵ cells/cm² in hPSC medium with Y-27632 (10 µM final concentration).
      2. Day 0–day 4: On d0, wash with DPBS and feed with RPMI with L-glutamine substitute (100x), FBS (0.5% final concentration), and Activin A (100 ng/mL final concentration). From d0, feed daily with the same media.
      3. Day 5: Fix with 4% paraformaldehyde and stain by immunohistochemistry for endoderm marker SOX17²¹ and confirm loss of pluripotency marker NANOG (Figure 4F).
         NOTE: Differentiations were done according to previously published protocols. In our experience matrix coatings can be interchangeable.

Results

Success of this protocol can be tracked at various stages. Table 1 offers a summary of samples on which this protocol has been performed. Starting samples, which are either cryopreserved buffy coats stored for one week or cryopreserved PBMCs, that have good sample quality upon thawing and the distinct morphological appearance after cell expansion successfully reprogrammed 6/7 times (Table 1, category 1). Two more buffy coat samples, collected and stored under similar conditions exhibited similar quality and morphology, but were taken for establishment of other assays, rather than reprogrammed. Alternatively, starting with frozen buffy coat that has been stored for long periods of time (> 6 years), regardless of their quality upon thawing, they will not exhibit the reprogrammable morphology after somatic cell expansion, and will not successfully reprogram (Table 1, category 2). To conserve resources, samples which do not exhibit the reprogrammable morphology after expansion should not be transduced (Table 1, category 3).

Overall successful reprogramming will depend on the health and wellbeing of the expanded PBMCs/blood cells, which is dependent on storage conditions. Immediately upon thawing, frozen blood samples may diffuse when added to DPBS (good), or dense droplets will sink directly to the bottom without diffusing (bad) which may signify that the cells have low viability (Figure 5 CK1).

Successful PBMC expansion will be marked by a distinct morphological appearance. PBMCs differentiated into erythroblasts appear as bright, round cells of large and small size at the end of the expansion phase (good), and these cells have shown to be the most successful to reprogram Cultures lacking both large and small bright round cells (bad), or that are dead, are unlikely to result in successful reprogramming (Figure 5 CK2).

Successful transduction and reprogramming of PBMCs will be marked by another distinct morphological change: the emergence of iPSC colonies on irradiated feeder cells. Colonies should begin to emerge 1–3 weeks following transduction. Emerging colonies will first look slightly morphologically different than the MEFs and will then grow to resemble a typical stem cell morphology (good). Emergence of cells that don’t adapt a stem cell like morphology (bad) are not a result of successful reprogramming and should not be carried forward (Figure 5 CK3). In the event that there is a morphological change, but not to the typical dense and round hPSC colony morphology, this is an indication that the cells are no longer PBMCs but have not been fully reprogrammed into iPSCs either.

Successfully reprogrammed iPSCs will exhibit distinct stem cell morphology, marked by dense, round colonies with smooth, bright edges (good). In the early passages after picking, subclones may have less ideal morphology with spontaneous differentiation or dense centers (OK), but adjusting splitting conditions can resolve these issues. Some picked colonies will not successfully expand (bad) (Figure 5 CK4). In addition to morphology, iPSCs should exhibit stemness as evidenced by expression of stemness markers (Figure 4C) such as OCT4, NANOG, or SOX2, and demonstrate differentiation potential into the three germ layers: ectoderm (Figure 4D), mesoderm (Figure 4E), and endoderm (Figure 4F).

Figure 1: Processed Blood Samples. (A) Frozen buffy coat after thawing and performing a density gradient centrifugation. The interface, marked by the arrowhead, contains the reprogrammable cells. (B) Frozen buffy coat after thawing. The cell pellet, marked by the arrowhead, contains the reprogrammable cells. (C) Frozen PBMCs after thawing. The cell pellet, marked by the arrowhead, contains the reprogrammable cells. Please click here to view a larger version of this figure.

Figure 2: PBMC expansion and morphology change into erythroblasts. (A) Expansion of PBMCs from samples prepared by direct thaw of buffy coat (row 1), density gradient performed after buffy coat is thawed (row 2), or thawed PBMCs, which were purified by density gradient before freezing (row 3). On the day of transduction, observe the presence of small round cells (blue arrows, PBMCs) and larger bright round cells (orange arrows, erythroblasts). (B) In some cases, after expansion, cells do not have the desired morphology, usually, lacking the large bright round cells. (C) Example of the morphology of dead cells. Scale bar represents 200 µm. Please click here to view a larger version of this figure.

Figure 3: Colony emergence and picking (A) Newly emerging colony 7 days after transduction. (B) Colony ready to be picked 14 days after transduction. (C) Abnormal morphology change, but not colony 14 days after transduction. Scale bar represents 200 µm. Please click here to view a larger version of this figure.

Figure 4: Expansion and characterization (A) Examples of good colonies (brightfield). (B) Examples of poor stem cell morphology include variable cell density within colonies, differentiation, or rough edges (red arrows). (C) Immunofluorescent image of undifferentiated, reprogrammed iPSCs 10 passages after transduction. Immunofluorescent image of reprogrammed iPSCs differentiated into (D) ectoderm (E) mesoderm, and (F) endoderm. Scale bar represents 200 µm. Please click here to view a larger version of this figure.

Figure 5: iPSC Reprogramming Checkpoints iPSC reprogramming from frozen buffy coats or PBMCs has four critical checkpoints that need to clear for success. (CK1) Checkpoint 1: patient sample quality upon thawing. (CK2) Checkpoint 2: Morphology after somatic cell expansion. (CK3) Checkpoint 3: Morphology as colonies emerge after transduction. (CK4) Checkpoint 4: iPSC morphology during characterization and expansion. Created in BioRender. Zeltner, N. (2025) https://BioRender.com/l70qwk4 Please click here to view a larger version of this figure.

Table 1: iPSC Reprogramming Sample Summary Please click here to download this Table.

Summary of all donor samples on which this protocol was used. CK1 refers to checkpoint 1, which assesses sample quality upon thawing. CK2 refers to checkpoint 2, which evaluates reprogrammable morphology after somatic cell expansion. FD denotes familial dysautonomia, CAH denotes congenital adrenal hyperplasia, and PTSD denotes post-traumatic stress disorder.

Category 1 includes buffy coats or PBMCs stored up to 1.5 years that show good sample quality upon thawing and reprogrammable morphology after transduction and are successfully reprogrammed. Category 2 includes buffy coat samples that appear to thaw well but do not adopt reprogrammable morphology after expansion and do not successfully reprogram. Category 3 includes samples that did not adopt reprogrammable morphology or did not survive expansion and were therefore not transduced in later experiments.

Discussion

iPSC technology has immense potential in the biomedical field with regard to disease mechanism discovery, personalized medicine, and “clinical trials in a dish”^22,23. Efficient and adaptable protocols for iPSC reprogramming from blood samples are key to unlocking the grand potential of iPSC technology as they are easy to collect and often readily available in biobanks. While there are many published protocols that detail reprogramming from purified PBMCs, this protocol details reprogramming from less purified blood samples i.e. frozen buffy coats and identifies checkpoints along the reprogramming timeline to easily gauge success, minimizing the waste of time and resources.

The first critical step for successful reprogramming is having high quality starting samples (Figure 5, CK1). PBMCs purified from fresh blood provide the most convenient starting sample for reprogramming; however, in this protocol, steps to obtain successful reprogramming starting from suboptimal samples such as frozen buffy coats are detailed. Purification by density gradient centrifugation upon thawing the buffy coat has shown to reduce contaminating cells, namely RBCs, and increase the chance of successful reprogramming. If the thawed buffy coat is extremely dense upon thawing (when dropping into DPBS, droplets retain their shape and sink directly to the bottom), this may indicate low cell viability and bad sample quality.

The next critical checkpoint in reprogramming is somatic cell morphology after expansion (Figure 5, CK2). Samples ready for transduction will have cells of large and small size that are round and bright. If after two weeks of this morphology is not seen, reprogramming success is unlikely.

Transduction and colony emergence are the next critical steps (Figure 5, CK3).

The final critical step in iPSC reprogramming is expansion and characterization (Figure 5, CK4). In the current protocol, characterization does not go beyond confirmation of pluripotency and differentiation potential. To further confirm that an iPSC line is suitable for use, it is important to demonstrate a normal karyotype and line stability upon continued passaging.

Although the troubleshooting described here can help lead to successful reprogramming, there are still limitations. Performing the density gradient causes significant cell loss, so in cases of low cell viability or otherwise suspected low cell counts, proceed to PBMC expansion without performing a density gradient. If the ideal morphology is not seen after somatic cell expansion, this may be due to issues with the quality of the starting sample. If after transduction, there are no emerging colonies, but there are cells with morphology distinct from either PBMCs or MEFs (Figure 3C), the cells have not successfully reprogrammed into iPSCs. At colony emergence, it is possible that colonies emerge but are lacking typical stem cell morphology. If colonies emerge but do not grow very large or start falling apart, pick them earlier, within a few days of emergence. If colonies grow with holes in the middle, like donuts, double check the growth factor concentrations and try adjusting the FGF2 concentration. Following colony emergence, it may be difficult to adapt picked colonies to your ideal culturing conditions. There is great variability between colonies, and it is not always possible to predict overall expansion success of individual colonies prior to picking, which is why it is recommended to pick many individual colonies and then pare down to a few successful clones. If no picked colonies are having success transitioning to feeder-free conditions, additional support may be necessary, such as a slower transition where colonies are picked and maintained in feeder conditions and more gradually transition to chemically defined and feeder-free conditions.

Even after picking and successful transition to preferred culturing conditions, reprogrammed lines may still exhibit poor stem cell morphology, or spontaneous differentiation. To combat these issues and clean up the lines, adjustments can be made during the splitting and maintenance process such as, splitting at a lower density or splitting with a shorter EDTA incubation time. If all else fails, colonies with good morphology can be picked as in step 7 above, to give a fresh start at clean lines.

The current protocol is one of the only to provide a detailed explanation of reprogramming from frozen buffy coats⁸, including critical checkpoints for monitoring and predicting reprogramming success. Notably, this protocol purifies frozen buffy coats using density gradient centrifugation after thawing, unlike previous protocols. Additionally, the detailing of the distinct reprogrammable morphology after somatic cell expansion is unique to this protocol and greatly enhances the accessibility of iPSC reprogramming from frozen buffy coats and PBMCs alike. Here, conventional PBMC reprogramming protocols⁹ are adapted for use with frozen buffy coats, unlocking the potential for reprogramming from many existing patient populations with existing biobanked samples which can allow for disease modeling without having to recruit new patients. Additionally, troubleshooting steps for working with sub-optimal starting samples or otherwise difficult to reprogram iPSC lines are detailed. Overall, this protocol can help stem cell researchers broaden the utility of iPSC technology.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 cm standard tissue culture dishes	Falcon	353003
12 well plates	Falcon	353043
15 mL conical tubes	Fisher	7200886
24 well plates	Corning	7200740
4% paraformaldehyde solution	Thermo Scientific	J19943-K2
6 well plates	Fisher Scientific	FB012927
accuSpin1R	Fisher Scientific	75003449
Activin A	Bio-techne	338-AC
AF488-donkey α goat	invitrogen	A11055
AF555-donkey α rbt	invitrogen	A31572
AF647-donkey α m	invitrogen	A31571
AF647-donkey α rbt	invitrogen	A31573
Antibiotic-Antimycotic (100X)	Fisher	15 240 096
Basix Polystyrene Serological Pipets	Fisher	14955233 14955234 14955235 14955236
BD Tuberculin Syringes	Fisher	14-826-87
BD Vacutaine Glass Mononuclear Cell Preparation (CPT) Tubes	Fisher Scientific	02-685-125	PBMC isolating blood collection tubes
BD Vacutainer Plastic Blood Collection Tubes with K2EDTA: Hemogard Closure	Fisher Scientific	02-657-32	anti-coagulent coated blood collection tubes
B-Mercaptoethanol	Fisher	21 985 023
Bovine Gelatin	Sigma	G1393
Brachyury-T antibody polyclonal goat IgG	R&D	AF2085
CellDrop BF	Denovix	CellDrop BF
Centrifuge 5702	Eppendorf	EP022628102-1EA
chemically defined lipid concentrate	Fisher	NC9022744
Chir 99021	R&D	4423
CO2 incubator	Sanyo	MCO-20AIC
Cryopreservation Storage System	Custom Biogenic Systems	V-1500AB
CryoStor CS10	Stem Cell Technologies	100-1061	alternate freezing medium
CytoTune-iPS 2.0 Sendai Reprogramming Kit	Fisher	A16517
Dexamethasone	Sigma	D2915
DMEM	Fisher	11965118
DMEM/F12	Fisher	11330057
DMSO	Fisher	BP231-1
DPBS 1X W/O CA MG	Fisher	MT21031CM
EDTA	Fisher	AM9262
Erythropoeitin (EPO)	Bio-Techne	287-TC-500
Essential 6 Medium (E6)	Fisher	A1516401	differentiation base
Essential 8 medium (E8)	Fisher	A1517001	hPSC basal medium
EVOS FL microscope	Thermo	AMF4300
Fetal Bovine Serum	VWR	45000-734
FGF basic (FGF2)	Bio-techne	233-FB/CF
Geltrex	Fisher	A1413202	Matrix coating
Gibco MEM Non-Essential Amino Acids Solution (100X)	Fisher	11140050
Glutamax	Fisher	35050061	L-glutamine substitute
Heparin	Stem Cell Technologies	7980
Histopaque 1077	Sigma	10771	density gradient solution 1
Holotransferrin	Sigma	T0665
Human Stem Cell Factor (SCF)	Peprotech	300-07-100UG
Insulin like Growth Factor 1 (IGF1)	Peprotech	100-11-100UG
Interleukin-3 (IL-3)	Peprotech	200-03-2UG
Isotemp 210 water bath	Fisher Scientific	210
Knockout-Serum-Replacement (KSR)	Fisher	10828028	FBS free supplement
L-Ascorbic Acid	Sigma	A4544-100G
LDN193189	Stemgent	04-0074
L-Glutamine	Fisher	25030081
Lymphoprep	Stem Cell Technologies	7801	Density gradient solution 2
Matrigel	Corning	CB-40234	Matrix coating
Mouse Embryonic Fibroblasts	Fisher	A34964
Nanog polyclonal goat IgG	R&D	AF1997-SP
Nunc Biobanking and Cell Culture Cryogenic Tubes	Fisher Scientific	12-565-163N
Penicillin/Streptomycin	Fisher	15140122
Purified anti-Pax-6 Antibody rabbit polyclonal IgG	Biolegend	901301
QBSF-60 Serum Free Medium	Quality Biological	160-204-101	EM base
Recovery Cell Culture Freezing Medium	Thermo Fisher Scientific	12648010	alternate freezing medium
rhVitronectin (VTN)	Fisher	A31804
RPMI 1640 Medium	Fisher	11875093
SB431542	R&D	1614
Sendai virus HN Monoclonal Antibody Mouse IgG2a	Invitrogen	14649482
Sodium Selenite	Sigma	S5261
Sox17 Antibody polyclonal goat IgG	R&D	af1924
StemSpan Erythroid Expansion supplement	Stem Cell Technologies	2692	Alternate commercial expansion medium supplement
StemSpan SFEM II medium	Stem Cell Technologies	9605	Alternate commercial expansion medium
Ultra Low Temperature Freezer	Thermo Fisher Scientific	TSX60086A
Universal Pipette Tips- Filtered	VWR	76322-132 76322-134 76322-150 76322-156
XAV939	Bio-techne	3748/10
Y-27632 Dihydrochloride	BioGems	1293823