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<article dtd-version="3.0" article-type="Invited Methods Article - JoVE Produced Video">
  <front>
    <journal-meta>
      <journal-id journal-id-type="publisher">jove</journal-id>
      <journal-id journal-id-type="email">editorial-office@jove.com</journal-id>
      <journal-title-group>
        <journal-title>Journal of Visualized Experiments</journal-title>
      </journal-title-group>
      <issn pub-type="ppub">1940-087X</issn>
      <issn pub-type="epub"></issn>
    </journal-meta>
    <article-meta>
      <article-id pub-id-type="publisher-id">JoVE52885</article-id>
      <article-id pub-id-type="manuscript">JoVE52885</article-id>
      <article-categories>
        <subj-group subj-group-type="Article Type">
          <subject>Invited Methods Article - JoVE Produced Video</subject>
        </subj-group>
        <subj-group subj-group-type="Category">
          <subject>JoVE Biology</subject>
        </subj-group>
        <subj-group subj-group-type="Classification">
          <subject>biology (general)</subject>
          <subject>Cells</subject>
          <subject>Biological Phenomena</subject>
        </subj-group>
      </article-categories>
      <title-group>
        <article-title>Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using non-integrating Episomal Plasmids</article-title>
      </title-group>
      <contrib-group>
        <contrib contrib-type="author">
          <role content-type="1" />
          <name>
            <surname>Meraviglia</surname>
            <given-names>Viviana</given-names>
          </name>
          <email>viviana.meraviglia@eurac.edu</email>
          <xref ref-type="aff" rid="aff1" />
        </contrib>
        <contrib contrib-type="author">
          <role content-type="2" />
          <name>
            <surname>Zanon</surname>
            <given-names>Alessandra</given-names>
          </name>
          <email>alessandra.zanon@eurac.edu</email>
          <xref ref-type="aff" rid="aff2" />
        </contrib>
        <contrib contrib-type="author">
          <role content-type="3" />
          <name>
            <surname>Alexandros</surname>
            <given-names>Lavdas A.</given-names>
          </name>
          <email>alexandros.lavdas@eurac.edu</email>
          <xref ref-type="aff" rid="aff3" />
        </contrib>
        <contrib contrib-type="author">
          <role content-type="4" />
          <name>
            <surname>Schwienbacher</surname>
            <given-names>Christine</given-names>
          </name>
          <email>christine.schwienbacher@eurac.edu</email>
          <xref ref-type="aff" rid="aff4" />
        </contrib>
        <contrib contrib-type="author">
          <role content-type="5" />
          <name>
            <surname>Silipigni</surname>
            <given-names>Rosamaria</given-names>
          </name>
          <email>rosamariasilipigni@gmail.com</email>
          <xref ref-type="aff" rid="aff5" />
        </contrib>
        <contrib contrib-type="author">
          <role content-type="6" />
          <name>
            <surname>Di Segni</surname>
            <given-names>Marina</given-names>
          </name>
          <email>marina.disegni@policlinico.mi.it</email>
          <xref ref-type="aff" rid="aff6" />
        </contrib>
        <contrib contrib-type="author">
          <role content-type="7" />
          <name>
            <surname>Chen</surname>
            <given-names>Huei-Sheng Vincent</given-names>
          </name>
          <email>hsv_chen@sanfordburnham.org</email>
          <xref ref-type="aff" rid="aff7" />
        </contrib>
        <contrib contrib-type="author">
          <role content-type="8" />
          <name>
            <surname>Pramstaller</surname>
            <given-names>Peter P.</given-names>
          </name>
          <email>peter.pramstaller@eurac.edu</email>
          <xref ref-type="aff" rid="aff8" />
        </contrib>
        <contrib contrib-type="author">
          <role content-type="9" />
          <name>
            <surname>Hicks</surname>
            <given-names>Andrew A.</given-names>
          </name>
          <email>andrew.hicks@eurac.edu</email>
          <xref ref-type="aff" rid="aff9" />
        </contrib>
        <contrib contrib-type="author" corresp="yes">
          <role content-type="10" />
          <name>
            <surname>Alessandra</surname>
            <given-names>Rossini</given-names>
            <prefix>Dr.</prefix>
          </name>
          <email>Alessandra.Rossini@eurac.edu</email>
          <xref ref-type="aff" rid="aff10" />
        </contrib>
        <contrib contrib-type="editor">
          <name>
            <surname>Nguyen</surname>
            <given-names>Nam</given-names>
          </name>
          <degrees>Ph.D.</degrees>
          <email>nam.nguyen@jove.com</email>
          <xref ref-type="aff" rid="aff11" />
        </contrib>
        <aff id="aff1">
          <institution>European Academy of Bozen-EURAC research</institution>
          <institution content-type="dept">Center for Biomedicine</institution>
          <addr-line content-type="addrline1">via Galvani 31</addr-line>
          <country>ITALY</country>
          <addr-line content-type="city">Bolzano</addr-line>
          <addr-line content-type="zipcode">39100</addr-line>
          <institution>European Academy of Bozen-EURAC research</institution>
        </aff>
        <aff id="aff2">
          <institution>European Academy of Bozen-EURAC research</institution>
          <institution content-type="dept">Center for Biomedicine</institution>
          <addr-line content-type="addrline1">via Galvani 31</addr-line>
          <country>ITALY</country>
          <addr-line content-type="city">Bolzano</addr-line>
          <addr-line content-type="zipcode">39100</addr-line>
          <institution>European Academy of Bozen-EURAC research</institution>
        </aff>
        <aff id="aff3">
          <institution>European Academy of Bozen-EURAC research</institution>
          <institution content-type="dept">Center for Biomedicine</institution>
          <addr-line content-type="addrline1">via Galvani 31</addr-line>
          <country>ITALY</country>
          <addr-line content-type="city">Bolzano</addr-line>
          <addr-line content-type="zipcode">39100</addr-line>
          <institution>European Academy of Bozen-EURAC research</institution>
        </aff>
        <aff id="aff4">
          <institution>European Academy of Bozen-EURAC research</institution>
          <institution content-type="dept">Center for Biomedicne</institution>
          <addr-line content-type="addrline1">via Galvani 31</addr-line>
          <country>ITALY</country>
          <addr-line content-type="city">Bolzano</addr-line>
          <addr-line content-type="zipcode">39100</addr-line>
          <institution>European Academy of Bozen-EURAC research</institution>
        </aff>
        <aff id="aff5">
          <institution>Ospedale Maggiore Policlinico</institution>
          <institution content-type="dept">Laboratory of Medical Genetics</institution>
          <addr-line content-type="addrline1">via Francesco Sforza 28</addr-line>
          <country>ITALY</country>
          <addr-line content-type="city">Milano</addr-line>
          <addr-line content-type="zipcode">20122</addr-line>
          <institution>Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico</institution>
        </aff>
        <aff id="aff6">
          <institution>Ospedale Maggiore Policlinico</institution>
          <institution content-type="dept">Laboratory of Medical Genetics</institution>
          <addr-line content-type="addrline1">via Francesco Sforza 28</addr-line>
          <country>ITALY</country>
          <addr-line content-type="city">Milano</addr-line>
          <addr-line content-type="zipcode">20122</addr-line>
          <institution>Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico</institution>
        </aff>
        <aff id="aff7">
          <institution>Sanford-Burnham Medical Research Institute</institution>
          <institution content-type="dept">Aging &amp; Stem Cell Research</institution>
          <addr-line content-type="addrline1">10901 N Torrey Pines Rd</addr-line>
          <country>UNITED STATES</country>
          <addr-line content-type="city">La Jolla</addr-line>
          <addr-line content-type="state">California</addr-line>
          <addr-line content-type="zipcode">92037</addr-line>
          <institution>Sanford-Burnham Medical Research Institute</institution>
        </aff>
        <aff id="aff8">
          <institution>European Academy of Bozen-EURAC research</institution>
          <institution content-type="dept">Center for Biomedicine</institution>
          <addr-line content-type="addrline1">via Galvani 31</addr-line>
          <country>ITALY</country>
          <addr-line content-type="city">Bolzano</addr-line>
          <addr-line content-type="zipcode">39100</addr-line>
          <institution>European Academy of Bozen-EURAC research</institution>
        </aff>
        <aff id="aff9">
          <institution>European Academy of Bozen-EURAC research</institution>
          <institution content-type="dept">Center for Biomedicine</institution>
          <addr-line content-type="addrline1">via Galvani 31</addr-line>
          <country>ITALY</country>
          <addr-line content-type="city">Bolzano</addr-line>
          <addr-line content-type="zipcode">39100</addr-line>
          <institution>European Academy of Bozen-EURAC research</institution>
        </aff>
        <aff id="aff10">
          <institution>European Academy of Bozen-EURAC research</institution>
          <institution content-type="position">PhD</institution>
          <institution content-type="dept">Center for Biomedicine</institution>
          <addr-line content-type="addrline1">via Galvani 31</addr-line>
          <country>ITALY</country>
          <addr-line content-type="city">Bolzano</addr-line>
          <addr-line content-type="state">Bolzano</addr-line>
          <addr-line content-type="zipcode">39100</addr-line>
          <phone content-type="primary">00390471055504</phone>
          <institution>European Academy of Bozen-EURAC research</institution>
        </aff>
        <aff id="aff11">
          <institution>JoVE, Journal of Visualized Experiments</institution>
          <addr-line>1 Alewife Center</addr-line>
          <addr-line>Suite 200</addr-line>
          <addr-line>Cambridge, MA  02140</addr-line>
          <country>UNITED STATES</country>
        </aff>
      </contrib-group>
      <history>
        <date date-type="received">
          <day>03</day>
          <month>11</month>
          <year>2014</year>
        </date>
        <date date-type="rev-recd">
          <day>18</day>
          <month>12</month>
          <year>2014</year>
        </date>
        <date date-type="rev-recd">
          <day>12</day>
          <month>11</month>
          <year>2014</year>
        </date>
        <date date-type="accepted">
          <day>18</day>
          <month>12</month>
          <year>2014</year>
        </date>
      </history>
      <abstract>
        <p>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 Ficoll separation.
</p>
      </abstract>
      <kwd-group>
        <kwd>stem cell biology</kwd>
        <kwd>cellular biology</kwd>
        <kwd>molecular biology</kwd>
        <kwd>induced pluripotent stem cells</kwd>
        <kwd>peripheral blood mononuclear cells</kwd>
        <kwd>reprogramming</kwd>
        <kwd>episomal plasmids.</kwd>
      </kwd-group>
      <funding-group />
      <counts>
        <fig-count count="0" />
      </counts>
      <custom-meta-group>
        <custom-meta>
          <meta-name>Please indicate the &lt;b&gt; full address&lt;/b&gt; at which this article will be &lt;b&gt;filmed&lt;/b&gt;.</meta-name>
          <meta-value>European Academy Bozen-EURAC Research-Center for Biomedicine via Galvani 31 39100 Bolzano ITALY</meta-value>
        </custom-meta>
        <custom-meta>
          <meta-name>If the invoicing address and point of contact are different than the corresponding author and filming address, please note the differences below.</meta-name>
          <meta-value>EURAC Research
Center for Biomedicine
via Druso 1
39100 Bolzano
ITALY</meta-value>
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        <custom-meta>
          <meta-name>Please indicate whether this article will be standard or open access. For information on the differences, &lt;a href="http://www.jove.com/publish/journal-information#7" target="_blank"&gt;click here&lt;/a&gt;</meta-name>
          <meta-value>Standard Access (US$2,400)</meta-value>
        </custom-meta>
        <custom-meta>
          <meta-name>If the protocol section of this manuscript is longer than 3 pages, are the most important steps highlighted (up to 2.75 pages)?&lt;/br&gt;
&lt;/br&gt;
This is &lt;b&gt;required &lt;/b&gt; for protocols longer than 3 pages, properly formatted (12 pt font, single spaced, space between each step, 1" margins, no indentations). Please see &lt;a href="http://www.jove.com/files/Instructions_for_Authors.pdf"&gt;JoVE Manuscript Instructions for Authors&lt;/a&gt; for more clarification. Remember that the non-highlighted protocol steps will remain in the manuscript and therefore will still be available to the reader. 
&lt;br/&gt;&lt;br/&gt;&lt;b&gt;Type 'Yes' or if needed, provide further explanation.&lt;/b&gt;</meta-name>
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          <meta-name>Please indicate the &lt;b&gt; full address&lt;/b&gt; at which this article will be &lt;b&gt;filmed&lt;/b&gt;.</meta-name>
          <meta-value>European Academy Bozen-EURAC Research-Center for Biomedicine via Galvani 31 39100 Bolzano ITALY</meta-value>
        </custom-meta>
        <custom-meta>
          <meta-name>If the invoicing address and point of contact are different than the corresponding author and filming address, please note the differences below.</meta-name>
          <meta-value>EURAC Research
Center for Biomedicine
via Druso 1
39100 Bolzano
ITALY</meta-value>
        </custom-meta>
        <custom-meta>
          <meta-name>Please indicate whether this article will be standard or open access. For information on the differences, &lt;a href="http://www.jove.com/publish/journal-information#7" target="_blank"&gt;click here&lt;/a&gt;</meta-name>
          <meta-value>Standard Access (US$2,400)</meta-value>
        </custom-meta>
        <custom-meta>
          <meta-name>If the protocol section of this manuscript is longer than 3 pages, are the most important steps highlighted (up to 2.75 pages)?&lt;/br&gt;
&lt;/br&gt;
This is &lt;b&gt;required &lt;/b&gt; for protocols longer than 3 pages, properly formatted (12 pt font, single spaced, space between each step, 1" margins, no indentations). Please see &lt;a href="http://www.jove.com/files/Instructions_for_Authors.pdf"&gt;JoVE Manuscript Instructions for Authors&lt;/a&gt; for more clarification. Remember that the non-highlighted protocol steps will remain in the manuscript and therefore will still be available to the reader. 
&lt;br/&gt;&lt;br/&gt;&lt;b&gt;Type 'Yes' or if needed, provide further explanation.&lt;/b&gt;</meta-name>
          <meta-value>Yes</meta-value>
        </custom-meta>
        <custom-meta>
          <meta-name>Is all of the written text in your manuscript original (not plagiarized, or self-plagiarized)?  
&lt;br&gt;&lt;br&gt;
&lt;em&gt;Exception-&lt;/em&gt; figure legends- if you are using previously published figures you must have permission to re-use the figure from the previous publisher and you must cite the original work in the figure legend.
&lt;br&gt;&lt;br&gt;
&lt;b&gt;Type 'Yes' or if needed, provide further explanation. &lt;/b&gt;</meta-name>
          <meta-value>Yes</meta-value>
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          <meta-name>Acquisitions Editor</meta-name>
          <meta-value>Nandita Singh</meta-value>
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          <meta-value>Jennifer Carroll</meta-value>
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      </custom-meta-group>
    </article-meta>
  </front>
  <body>
    <table-wrap>
      <label>Table 1</label>
      <graphic xlink:href="Primer list.xlsx" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </table-wrap>
    <table-wrap>
      <label>Excel Spreadsheet- Table of Materials/Equipment</label>
      <graphic xlink:href="Copy of JoVE_Materials DEF.xlsx" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </table-wrap>
    <fig>
      <label>Figure 2</label>
      <graphic xlink:href="Figure 2.pdf" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </fig>
    <fig>
      <label>Figure 3</label>
      <graphic xlink:href="Figure 3.pdf" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </fig>
    <fig>
      <label>Figure 4</label>
      <graphic xlink:href="Figure 4.pdf" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </fig>
    <fig>
      <label>Figure 5</label>
      <graphic xlink:href="Figure 5.pdf" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </fig>
    <fig>
      <label>Figure 6</label>
      <graphic xlink:href="Figure 6.pdf" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </fig>
    <fig>
      <label>Figure 1</label>
      <graphic xlink:href="Figure 1_revised.pdf" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </fig>
    <fig>
      <label>Figure 2</label>
      <graphic xlink:href="Figure 2_revised.pdf" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </fig>
    <fig>
      <label>Figure 3</label>
      <graphic xlink:href="Figure 3_revised.pdf" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </fig>
    <fig>
      <label>Figure 4</label>
      <graphic xlink:href="Figure 4_revised.pdf" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </fig>
    <fig>
      <label>Figure 5</label>
      <graphic xlink:href="Figure 5_revised.pdf" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </fig>
    <fig>
      <label>Figure 6</label>
      <graphic xlink:href="Figure 6_revised.pdf" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </fig>
    <fig>
      <label>Figure 1</label>
      <graphic xlink:href="Figure 1.pdf" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </fig>
  </body>
</article>