The study was supported by the Ministry of Health and Department of Educational Assistance, University and Research of the Autonomous Province of Bolzano and the South Tyrolean Sparkasse Foundation.

Peripheral blood mononuclear cells (PBMNCs) were isolated from human peripheral blood samples of healthy donors after signed informed consent and approval of the Ethical Committee of the Province of South Tyrol. Experiments were conducted in accordance with the principles expressed in the Declaration of Helsinki. All data were collected and analyzed anonymously.
1. Isolation of Peripheral Blood Mononuclear Cells (PBMNCs)
<ol>
	<li>PBMNCs obtained from buffy coats after whole blood centrifuga...

<ol>
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In the past, the only way to obtain human pluripotent stem cells carrying a particular genetic mutation was to recruit parents undergoing pre-implantation genetic diagnosis and generate embryonic stem cells from their discarded blastocysts31,32. Using a reprogramming approach, researchers can now generate iPSCs from patients carrying virtually any genotype. The possibility to start from a patient-specific cell line and an easily accessible source is very important since it allows an investigation of the pathop...

In 2006, the group of Shinya Yamanaka1 demonstrated for the first time that somatic cells from adult mice and humans can be converted into a pluripotent state by ectopic expression of four reprogramming factors (Oct-4, Sox-2, Klf-4, and c-Myc), generating the so-called Induced Pluripotent Stem Cells (iPSCs)2. Patient-specific iPSCs closely resemble human embryonic stem cells (hESCs) in terms of morphology, proliferation and ability to differentiate into the three-germ cell types (mesoderm, endoderm and ectoderm) while lacking the ethical concerns associated to the use of hESCs and bypassing possible immune rejection3. Thus, iPSCs appear as one of the most important sources of patient-specific cells for basic research, drug screening, disease modeling, evaluation of toxicity, and regenerative medicine purposes4.
Several approaches have been used for iPSC generation: viral integrating vectors (retrovirus5, lentivirus6), viral non-integrating vectors (adenovirus7), Sendai-virus8, BAC transposons9, episomal vectors10, proteins11 or RNA delivery12. Although the use of virus-mediated methods can lead to high efficiency reprogramming, viral vectors integrate into the genome of host cells and therefore potential random insertional mutagenesis, permanent alteration of gene expression, and reactivation of silenced transgenes during differentiation cannot be excluded13.
To make iPSCs safer for regenerative medicine, efforts have been made to derive iPSCs without the integration of exogenous DNA into cellular genomes. Although excisable viral vectors and transposons have been developed, it is still unclear whether short vector sequences, which inevitably remain in the transduced cells after excision, and transposase expression, could induce alteration in cellular function13. Despite its high reprogramming efficiency, Sendai virus represents an expensive approach and reach-through licensing concerns with the company that developed this system have the potential to limit its application in translational studies. Furthermore, the need for direct introduction of proteins and RNA requires multiple delivery of reprogramming molecules with the inherent technical limitations this introduces, and overall reprogramming efficiency is very low14. Of note, cost-effective viral-free and non-integrating methods based on the use of episomal plasmids have been successfully reported for the reprogramming of skin fibroblasts15. Specifically, in the present work we decided to use commercial available integration-free episomal plasmids, as previously reported10,15.
To date, skin fibroblasts represent the most popular donor cell type5. However, other cell sources have been successfully reprogrammed into iPSCs including keratinocytes16, bone marrow mesenchymal stem cells17, adipose stromal cells18, hair follicles19, and dental pulp cells20. The isolation of these cells requires surgical procedures, and several weeks are needed for in vitro cell expansion in order to establish a primary cell culture.
In this light, the selection of starting cell type is critical and it is equally important to be able to produce iPSCs from easily accessible and less invasive tissues such as blood. Both cord blood mononuclear cells (CBMNCs)21,22 and peripheral blood mononuclear cells (PBMNCs)14,22-24 represent suitable sources of cells for the derivation of iPSCs.
Although the efficiency of adult PBMNC reprogramming is 20&#8211;50 times lower than that of CBMNCs22, they remain the most convenient cell type for sampling purpose. In fact, PBMNC sampling has the advantage of being minimally invasive, and in addition, these cells do not require extensive expansion in vitro before reprogramming experiments. To date, different protocols have reported that PBMNCs after density gradient separation can be frozen and thawed days to several months after freezing and expanded for few days before reprogramming into iPSCs22,23. Nevertheless, as far as we are aware no reports have described reprogramming of PBMNCs from frozen buffy coats. Importantly, frozen buffy coats collected without density gradient separation represent the most common blood samples stored in large scale biobanks from population studies, thus representing an easily accessible pool of material for iPSC production that avoids further sample collection.
Herein we report for the first time the generation of viral-free iPSCs from human frozen buffy coats, based on a previously described protocol22. In addition, iPSCs were generated from frozen PBMNCs obtained after density gradient separation, as a control protocol for the non-density gradient purified PBMNC results.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
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			<td>Sodium Citrate buffered Venosafe Plastic Tube</td>
			<td>Terumo</td>
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			<td>Sigma-Aldrich</td>
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			<td>Sigma-Aldrich</td>
			<td>60339</td>
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			<td>Sigma-Aldrich</td>
			<td>E5134</td>
			<td>0.5 M solution</td>
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			<td>Ficoll-Paque Premium&#160;</td>
			<td>GE Healthcare Life Sciences</td>
			<td>17-5442-02</td>
			<td>Polysucrose solution for density gradient centrifugation</td>
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			<td>Iscove&#39;s modified Dulbecco&#39;s medium (IMDM)&#160;</td>
			<td>Gibco</td>
			<td>21056-023</td>
			<td>No phenol red</td>
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		<tr>
			<td>Ham&#39;s F-12</td>
			<td>Mediatech</td>
			<td>10-080-CV</td>
			<td></td>
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			<td>Insulin-Transferrin-Selenium-Ethanolamine (ITS -X)</td>
			<td>Gibco</td>
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			<td>1X (Stock: 100X)</td>
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			<td>Chemically Defined Lipid Concentrate</td>
			<td>Gibco</td>
			<td>11905031</td>
			<td>1X (Stock: 100X)</td>
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			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A9418</td>
			<td></td>
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			<td>L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>A8960</td>
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			<td>1-Thioglycerol</td>
			<td>Sigma-Aldrich</td>
			<td>M6145</td>
			<td>Final Concentration at 200 &#181;M</td>
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		<tr>
			<td>Recombinant Human Stem Cell Factor (SCF)</td>
			<td>PeproTech</td>
			<td>300-07</td>
			<td>100 ng/ml (Stock:100 &#181;g/ml)&#160;</td>
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			<td>PeproTech</td>
			<td>200-03</td>
			<td>10 ng/ml (Stock: 10 &#181;g/ml)</td>
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			<td>PeproTech</td>
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			<td>40 ng/ml (Stock: 40 &#181;g/ml)</td>
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			<td>R&#38;D Systems&#160;</td>
			<td>287-TC-500</td>
			<td>2 U/ml (Stock: 50 U/ml)</td>
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			<td>Sigma-Aldrich</td>
			<td>D2915</td>
			<td>1 &#956;M (Stock: 1 mM)</td>
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			<td>Human Holo-Transferrin</td>
			<td>R&#38;D Systems&#160;</td>
			<td>2914-HT</td>
			<td>100 &#956;g/ml (Stock: 20 mg/ml)</td>
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			<td>Amniomax II</td>
			<td>Gibco</td>
			<td>11269016</td>
			<td>Medium for cytogenetic analysis</td>
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			<td>mTeSR1</td>
			<td>StemCell Technologies</td>
			<td>5850</td>
			<td>Medium for iPSC feeder-free culture</td>
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		<tr>
			<td>Knockout DMEM</td>
			<td>Gibco</td>
			<td>10829-018</td>
			<td></td>
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			<td>Knockout Serum Replacement</td>
			<td>Gibco</td>
			<td>10828-028</td>
			<td></td>
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		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol&#160;</td>
			<td>Gibco</td>
			<td>31350-010</td>
			<td>0.1 mM (Stock: 50 mM)</td>
		</tr>
		<tr>
			<td>Sodium Butyrate</td>
			<td>Sigma-Aldrich</td>
			<td>B5887</td>
			<td>0.25 mM (Stock: 0.5 M)</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF basic, 145 aa&#160;</td>
			<td>R&#38;D Systems&#160;</td>
			<td>4114-TC</td>
			<td>10 ng/ml (Stock: 10 &#181;g/ml)</td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>Y0503&#160;</td>
			<td>10 &#181;M (Stock: 10 mM)</td>
		</tr>
		<tr>
			<td>Fetal Defined Bovine Serum</td>
			<td>Hyclone</td>
			<td>SH 30070.03</td>
			<td></td>
		</tr>
		<tr>
			<td>EmbryoMax 0.1% Gelatin Solution</td>
			<td>Merck-Millipore</td>
			<td>ES-006-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Basement Membrane Matrix Growth Factor Reduced</td>
			<td>BD Biosciences</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase, Type IV</td>
			<td>Gibco</td>
			<td>17104-019</td>
			<td>1 mg/ml (Stock: 10 mg/ml)</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>PAA Laboratories GmbH</td>
			<td>L11-007</td>
			<td>Cell detachment solution</td>
		</tr>
		<tr>
			<td>Mouse Embryonic Fibroblast (CF1)</td>
			<td>Global Stem</td>
			<td>GSC-6201G</td>
			<td>1*106 cells/6 well plate</td>
		</tr>
		<tr>
			<td>Plasmid pCXLE-hOCT3/4-shp53-F</td>
			<td>Addgene&#160;</td>
			<td>27077</td>
			<td>1 &#181;g (Stock: 1 &#181;g/&#181;l)</td>
		</tr>
		<tr>
			<td>Plasmid pCXLE-hSK</td>
			<td>Addgene&#160;</td>
			<td>27078</td>
			<td>1 &#181;g (Stock: 1 &#181;g/&#181;l)</td>
		</tr>
		<tr>
			<td>Plasmid pCXLE-hUL</td>
			<td>Addgene&#160;</td>
			<td>27080</td>
			<td>1 &#181;g (Stock: 1 &#181;g/&#181;l)</td>
		</tr>
		<tr>
			<td>Plasmid pCXLE-EGFP</td>
			<td>Addgene&#160;</td>
			<td>27082</td>
			<td>1 &#181;g (Stock: 1 &#181;g/&#181;l)</td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase Staining Kit</td>
			<td>Stemgent</td>
			<td>00-0009</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Stage-Specific Embryonic Antigen-4 (SSEA-4) Antibody</td>
			<td>Merck-Millipore</td>
			<td>MAB4304</td>
			<td>1/250</td>
		</tr>
		<tr>
			<td>Anti-TRA-1-60 Antibody</td>
			<td>Merck-Millipore</td>
			<td>MAB4360</td>
			<td>1/250</td>
		</tr>
		<tr>
			<td>Anti-TRA-1-81 Antibody</td>
			<td>Merck-Millipore</td>
			<td>MAB4381</td>
			<td>1/250</td>
		</tr>
		<tr>
			<td>Anti-Oct-3/4 Antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-9081</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Anti-Nanog Antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-33759</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Anti-Troponin I Antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-15368</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Anti-&#945;-Actinin (Sarcomeric) Antibody</td>
			<td>Sigma-Aldrich</td>
			<td>A7732</td>
			<td>1/250</td>
		</tr>
		<tr>
			<td>Neuronal Class III &#223;-Tubulin (TUJ1) Antibody</td>
			<td>Covance Research Products Inc&#160;</td>
			<td>MMS-435P-100</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Anti-Tyrosine Hydroxylase (TH) Antibody</td>
			<td>Calbiochem</td>
			<td>657012</td>
			<td>1/200</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat Anti-Mouse&#160;</td>
			<td>Molecular Probes</td>
			<td>A-11029</td>
			<td>1/1000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 Goat Anti-Rabbit</td>
			<td>Molecular Probes</td>
			<td>A-21429</td>
			<td>1/1000</td>
		</tr>
		<tr>
			<td>Ultra-Low Attachment Cell Culture 6-well plate</td>
			<td>Corning</td>
			<td>3471</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol Reagent</td>
			<td>Ambion</td>
			<td>15596-018&#160;</td>
			<td>reagent for RNA extraction</td>
		</tr>
		<tr>
			<td>SuperScript VILO cDNA Synthesis Kit</td>
			<td>Invitrogen</td>
			<td>11754050</td>
			<td>Reverse transcriptase kit</td>
		</tr>
		<tr>
			<td>iTaq Universal SYBR Green Supermix</td>
			<td>Bio-Rad</td>
			<td>172-5124</td>
			<td></td>
		</tr>
		<tr>
			<td>CFX96 Real-Time PCR Detection System</td>
			<td>Bio-Rad</td>
			<td>185-5195</td>
			<td></td>
		</tr>
		<tr>
			<td>Experion Automated Electrophoresis System</td>
			<td>Bio-Rad</td>
			<td>700-7000</td>
			<td>Instrument to check RNA integrity</td>
		</tr>
		<tr>
			<td>Experion RNA Highsense Analysis kit</td>
			<td>Bio-Rad</td>
			<td>7007105</td>
			<td>Reagent kit to check RNA integrity</td>
		</tr>
		<tr>
			<td>Dissecting microscope (SteREO Discovery V12 )</td>
			<td>Zeiss</td>
			<td>495007</td>
			<td></td>
		</tr>
		<tr>
			<td>NeonTransfection System 100 &#181;L Kit</td>
			<td>Invitrogen</td>
			<td>MPK10025</td>
			<td>Reagent kit for electroporation</td>
		</tr>
		<tr>
			<td>Neon Transfection System</td>
			<td>Invitrogen</td>
			<td>MPK5000</td>
			<td>Instrument used for electroporation</td>
		</tr>
		<tr>
			<td>NanoDrop UV/Vis Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>ND-2000</td>
			<td>Instrument for DNA/RNA quantification</td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>Qiagen</td>
			<td>12362</td>
			<td>Plasmid purification kit</td>
		</tr>
	</tbody>
</table>

generation of induced pluripotent stem cells from frozen buffy coats using non-integrating episomal plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

In this study a simple and effective protocol for the generation of viral-free iPSCs by reprogramming of PBMNCs isolated from frozen buffy coats after whole blood centrifugation and comparison with reprogramming of PBMNCs obtained after density gradient separation is reported. Figure 1A shows a schematic representation of the detailed protocol. After thawing, the isolated PBMNCs, showing a typical rounded shape, are expanded in specific blood culture medium for 14 days (Figure 1B) and th...

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids at JoVE.com

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

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.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

generation-induced-pluripotent-stem-cells-from-frozen-buffy-coats

Research

JoVE Journal

Developmental Biology

12.8K ビュー.  European Academy Bozen/Bolzano (EURAC). 誘導多能性幹細胞（iPS細胞）が臨床応用および基礎研究のための患者特有の組織のソースを表します。ここでは、非組み込みエピソームプラスミドを用いてウイルスを含まないiPS細胞に凍結バフィーコートから得たヒト末梢血単核細胞（PBMNCs）を再プログラムするために詳細なプロトコルを提示します。 