Name: Reprogramming Induced Pluripotent Stem Cell Lines from Frozen Buffy Coat Samples (Video) | JoVE Journal
Uploaded: 2026-04-10T12:50:00.000+00:00
Duration: 9 min 29 s
Description: Learn to reprogram frozen buffy coats and PBMCs into iPSCs using Sendai virus with Yamanaka factors. This protocol includes purification steps, morphological checkpoints, and troubleshooting for biobanked samples.

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Human subjects were recruited and provided informed consent for blood donation according to the IRB protocol UGA# 2012-10769-06, NYU# s21-01701, Emory# 2024P008659, and Emory# 000002114. Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institute of Health under award number 5T32GM142623 to J. Art, R01MH115174 to V. Michopoulos, and R01NS114567 to N. Zeltner. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

<ol><li>Takahashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131 (5), 861-872 (2007).</li>
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<li>Perry, J. N., Jasim, A., Hojat, A., Yong, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Procurement%2C+storage%2C+and+use+of+blood+in+biobanks%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Procurement, storage, and use of blood in biobanks.</a> Methods Mol Biol. 1897, 89-97 (2019).</li>
<li>Meraviglia, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+induced+pluripotent+stem+cells+from+frozen+buffy+coats+using+non-integrating+episomal+plasmids%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Generation of induced pluripotent stem cells from frozen buffy coats using non-integrating episomal plasmids.</a> J Vis Exp. (100), e52885(2015).</li>
<li>Yang, W., et al. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aYang%2C+W+%22iPSC+reprogramming+from+human+peripheral+blood+using+Sendai+Virus+mediated+gene+transfer%22">iPSC reprogramming from human peripheral blood using Sendai Virus mediated gene transfer</a>. , Stembook. Cambridge (MA). (2008).</li>
<li>Cerneckis, J., Cai, H., Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induced+pluripotent+stem+cells+%28ipscs%29%3A+Molecular+mechanisms+of+induction+and+applications%5BTitle%5D)+AND+%22Signal+Transduct+Target+Ther%22%5BJournal%5D)">Induced pluripotent stem cells (ipscs): Molecular mechanisms of induction and applications.</a> Signal Transduct Target Ther. 9 (1), 112(2024).</li>
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<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22guidelines+on+drawing+blood%3A+best+practices+in+phlebotomy%22">guidelines on drawing blood: best practices in phlebotomy</a>. , World Health Organization. Geneva. (2010).</li>
<li>Robb, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytokine+receptors+and+hematopoietic+differentiation%5BTitle%5D)+AND+%22Oncogene%22%5BJournal%5D)">Cytokine receptors and hematopoietic differentiation.</a> Oncogene. 26 (47), 6715-6723 (2007).</li>
<li>Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Trypan+blue+exclusion+test+of+cell+viability%5BTitle%5D)+AND+%22Curr+Protoc+Immunol%22%5BJournal%5D)">Trypan blue exclusion test of cell viability.</a> Curr Protoc Immunol. 111, A3 B 1-A3 B 3 (2015).</li>
<li>Chen, G. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aChen%2C+G+%22Splitting+hESC%2FhiPSC+lines+with+EDTA+in+feeder+free+conditions%22">Splitting hESC/hiPSC lines with EDTA in feeder free conditions</a>. , Stembook. Cambridge (MA). (2008).</li>
<li>Zeltner, N., Lafaille, F. G., Fattahi, F., Studer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Feeder-free+derivation+of+neural+crest+progenitor+cells+from+human+pluripotent+stem+cells%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Feeder-free derivation of neural crest progenitor cells from human pluripotent stem cells.</a> J Vis Exp. (87), e51609(2014).</li>
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<li>Lin, Y., Zou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differentiation+of+cardiomyocytes+from+human+pluripotent+stem+cells+in+fully+chemically+defined+conditions%5BTitle%5D)+AND+%22STAR+Protoc%22%5BJournal%5D)">Differentiation of cardiomyocytes from human pluripotent stem cells in fully chemically defined conditions.</a> STAR Protoc. 1 (1), (2020).</li>
<li>Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brachyury%2C+the+blastopore+and+the+evolution+of+the+mesoderm%5BTitle%5D)+AND+%22Bioessays%22%5BJournal%5D)">Brachyury, the blastopore and the evolution of the mesoderm.</a> Bioessays. 23 (9), 788-794 (2001).</li>
<li>Zeltner, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Capturing+the+biology+of+disease+severity+in+a+psc-based+model+of+familial+dysautonomia%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">Capturing the biology of disease severity in a psc-based model of familial dysautonomia.</a> Nat Med. 22 (12), 1421-1427 (2016).</li>
<li>Kanai-Azuma, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Depletion+of+definitive+gut+endoderm+in+sox17-null+mutant+mice%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Depletion of definitive gut endoderm in sox17-null mutant mice.</a> Development. 129 (10), 2367-2379 (2002).</li>
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We declare that the research was conducted without commercial or financial relationships that could be construed as a potential conflict of interest.

iPSC technology has immense potential in the biomedical field with regard to disease mechanism discovery, personalized medicine, and &#8220;clinical trials in a dish&#8221;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 PB...

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 cells2,3, each with their own limitations, are some of the most frequently used somatic cell sources&#160;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 protocols1,8,9 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&#8217;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 protocols8,9, 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&#8211;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 &#60; 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.

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

reprogramming induced pluripotent stem cell lines from frozen buffy coat samples

Reprogramming Induced Pluripotent Stem Cell Lines from Frozen Buffy Coat Samples

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.

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...

The current protocol details steps for troubleshooting induced pluripotent stem cell (iPSC) derivation from patient samples&#8217; peripheral blood mononuclear cells (PBMCs) that have been difficult to reprogram following the manufacturer&#8217;s instructions. This protocol is specific to frozen buffy coat samples but may also be applied to the reprogramming of purified PBMCs.

Human pluripotent stem cells (hPSCs) are a valuable tool for disease modeling. Further stem cells can be reprogrammed from adult somatic cells, called ...

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.

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Research

JoVE Journal

Biology

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Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

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We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

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Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

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Learn to reprogram frozen buffy coats and PBMCs into iPSCs using Sendai virus with Yamanaka factors. This protocol includes purification steps, morphological checkpoints, and troubleshooting for biobanked samples.

Reprogramming Induced Pluripotent Stem Cell Lines from Frozen Buffy Coat Samples

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