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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Early reprogramming regulators identified by prospective isolation and mass cytometry</title><author><name sortKey="Lujan, Ernesto" sort="Lujan, Ernesto" uniqKey="Lujan E" first="Ernesto" last="Lujan">Ernesto Lujan</name><affiliation><nlm:aff id="A1">Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA 94305, USA</nlm:aff></affiliation><affiliation><nlm:aff id="A2">Department of Genetics, Stanford University, Stanford, CA 94305, USA</nlm:aff></affiliation><affiliation><nlm:aff id="A4">Department of Pathology, Stanford University, Stanford, CA 94305, USA</nlm:aff></affiliation></author><author><name sortKey="Zunder, Eli R" sort="Zunder, Eli R" uniqKey="Zunder E" first="Eli R." last="Zunder">Eli R. Zunder</name><affiliation><nlm:aff id="A3">Baxter Laboratory in Stem Cell Biology, Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA</nlm:aff></affiliation></author><author><name sortKey="Ng, Yi Han" sort="Ng, Yi Han" uniqKey="Ng Y" first="Yi Han" last="Ng">Yi Han Ng</name><affiliation><nlm:aff id="A1">Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA 94305, USA</nlm:aff></affiliation><affiliation><nlm:aff id="A4">Department of Pathology, Stanford University, Stanford, CA 94305, USA</nlm:aff></affiliation><affiliation><nlm:aff id="A5">Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA</nlm:aff></affiliation></author><author><name sortKey="Goronzy, Isabel N" sort="Goronzy, Isabel N" uniqKey="Goronzy I" first="Isabel N." last="Goronzy">Isabel N. Goronzy</name><affiliation><nlm:aff id="A1">Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA 94305, USA</nlm:aff></affiliation><affiliation><nlm:aff id="A4">Department of Pathology, Stanford University, Stanford, CA 94305, USA</nlm:aff></affiliation></author><author><name sortKey="Nolan, Garry P" sort="Nolan, Garry P" uniqKey="Nolan G" first="Garry P." last="Nolan">Garry P. Nolan</name><affiliation><nlm:aff id="A3">Baxter Laboratory in Stem Cell Biology, Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA</nlm:aff></affiliation></author><author><name sortKey="Wernig, Marius" sort="Wernig, Marius" uniqKey="Wernig M" first="Marius" last="Wernig">Marius Wernig</name><affiliation><nlm:aff id="A1">Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA 94305, USA</nlm:aff></affiliation><affiliation><nlm:aff id="A4">Department of Pathology, Stanford University, Stanford, CA 94305, USA</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">25830878</idno><idno type="pmc">4441548</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4441548</idno><idno type="RBID">PMC:4441548</idno><idno type="doi">10.1038/nature14274</idno><date when="2015">2015</date><idno type="wicri:Area/Pmc/Corpus">000398</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000398</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Early reprogramming regulators identified by prospective isolation and mass cytometry</title><author><name sortKey="Lujan, Ernesto" sort="Lujan, Ernesto" uniqKey="Lujan E" first="Ernesto" last="Lujan">Ernesto Lujan</name><affiliation><nlm:aff id="A1">Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA 94305, USA</nlm:aff></affiliation><affiliation><nlm:aff id="A2">Department of Genetics, Stanford University, Stanford, CA 94305, USA</nlm:aff></affiliation><affiliation><nlm:aff id="A4">Department of Pathology, Stanford University, Stanford, CA 94305, USA</nlm:aff></affiliation></author><author><name sortKey="Zunder, Eli R" sort="Zunder, Eli R" uniqKey="Zunder E" first="Eli R." last="Zunder">Eli R. Zunder</name><affiliation><nlm:aff id="A3">Baxter Laboratory in Stem Cell Biology, Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA</nlm:aff></affiliation></author><author><name sortKey="Ng, Yi Han" sort="Ng, Yi Han" uniqKey="Ng Y" first="Yi Han" last="Ng">Yi Han Ng</name><affiliation><nlm:aff id="A1">Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA 94305, USA</nlm:aff></affiliation><affiliation><nlm:aff id="A4">Department of Pathology, Stanford University, Stanford, CA 94305, USA</nlm:aff></affiliation><affiliation><nlm:aff id="A5">Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA</nlm:aff></affiliation></author><author><name sortKey="Goronzy, Isabel N" sort="Goronzy, Isabel N" uniqKey="Goronzy I" first="Isabel N." last="Goronzy">Isabel N. Goronzy</name><affiliation><nlm:aff id="A1">Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA 94305, USA</nlm:aff></affiliation><affiliation><nlm:aff id="A4">Department of Pathology, Stanford University, Stanford, CA 94305, USA</nlm:aff></affiliation></author><author><name sortKey="Nolan, Garry P" sort="Nolan, Garry P" uniqKey="Nolan G" first="Garry P." last="Nolan">Garry P. Nolan</name><affiliation><nlm:aff id="A3">Baxter Laboratory in Stem Cell Biology, Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA</nlm:aff></affiliation></author><author><name sortKey="Wernig, Marius" sort="Wernig, Marius" uniqKey="Wernig M" first="Marius" last="Wernig">Marius Wernig</name><affiliation><nlm:aff id="A1">Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA 94305, USA</nlm:aff></affiliation><affiliation><nlm:aff id="A4">Department of Pathology, Stanford University, Stanford, CA 94305, USA</nlm:aff></affiliation></author></analytic><series><title level="j">Nature</title><idno type="ISSN">0028-0836</idno><idno type="eISSN">1476-4687</idno><imprint><date when="2015">2015</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p id="P2">In the context of most induced pluripotent stem (iPS) cell reprogramming methods, heterogeneous populations of nonproductive and staggered productive intermediates arise at different reprogramming time points<sup><xref rid="R1" ref-type="bibr">1</xref>–<xref rid="R11" ref-type="bibr">11</xref></sup>. Despite recent reports claiming substantially increased reprogramming efficiencies using genetically modified donor cells<sup><xref rid="R12" ref-type="bibr">12</xref>,<xref rid="R13" ref-type="bibr">13</xref></sup> prospectively isolating distinct reprogramming intermediates remains an important goal to decipher reprogramming mechanisms. Previous attempts to identify surface markers of intermediate cell populations were based on the assumption that during reprogramming cells progressively lose donor cell identity and gradually acquire iPS cell properties<sup><xref rid="R1" ref-type="bibr">1</xref>,<xref rid="R2" ref-type="bibr">2</xref>,<xref rid="R7" ref-type="bibr">7</xref>,<xref rid="R8" ref-type="bibr">8</xref>,<xref rid="R10" ref-type="bibr">10</xref></sup>. Here, we report that iPS cell and epithelial markers, such as SSEA1 and EpCAM, respectively, are not predictive of reprogramming during early phases. Instead, in a systematic functional surface marker screen we find that early reprogramming-prone cells express a unique set of surface markers, including CD73, CD49d and CD200 that are absent in fibroblasts and iPS cells. Single cell mass cytometry and prospective isolation show that these distinct intermediates are transient and bridge the gap between donor cell silencing and pluripotency marker acquisition during the early, presumably stochastic reprogramming phase<sup><xref rid="R2" ref-type="bibr">2</xref></sup>. Expression profiling revealed early upregulation of the transcriptional regulators <italic>Nr0b1</italic> and <italic>Etv5</italic> in this reprogramming state, preceding activation of key pluripotency regulators such as <italic>Rex1, Dppa2, Nanog</italic> and <italic>Sox2</italic>. Both factors are required for the generation of the early intermediate state and fully reprogrammed iPS cells, and thus mark some of the earliest known regulators of iPS cell induction. Our study deconvolutes the first steps in a hierarchical series of events that lead to pluripotency acquisition.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Brambrink, T" uniqKey="Brambrink T">T Brambrink</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Buganim, Y" uniqKey="Buganim Y">Y Buganim</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Golipour, A" uniqKey="Golipour A">A Golipour</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chen, J" uniqKey="Chen J">J Chen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hanna, J" uniqKey="Hanna J">J Hanna</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ho, R" uniqKey="Ho R">R Ho</name></author><author><name sortKey="Papp, B" uniqKey="Papp B">B Papp</name></author><author><name sortKey="Hoffman, Ja" uniqKey="Hoffman J">JA Hoffman</name></author><author><name sortKey="Merrill, Bj" uniqKey="Merrill B">BJ Merrill</name></author><author><name sortKey="Plath, K" uniqKey="Plath K">K Plath</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="O Malley, J" uniqKey="O Malley J">J O'Malley</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Polo, Jm" uniqKey="Polo J">JM Polo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sridharan, R" uniqKey="Sridharan R">R Sridharan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Stadtfeld, M" uniqKey="Stadtfeld M">M Stadtfeld</name></author><author><name sortKey="Maherali, N" uniqKey="Maherali N">N Maherali</name></author><author><name sortKey="Breault, Dt" uniqKey="Breault D">DT Breault</name></author><author><name sortKey="Hochedlinger, K" uniqKey="Hochedlinger K">K Hochedlinger</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tanabe, K" uniqKey="Tanabe K">K Tanabe</name></author><author><name sortKey="Nakamura, M" uniqKey="Nakamura M">M Nakamura</name></author><author><name sortKey="Narita, M" uniqKey="Narita M">M Narita</name></author><author><name sortKey="Takahashi, K" uniqKey="Takahashi K">K Takahashi</name></author><author><name sortKey="Yamanaka, S" uniqKey="Yamanaka S">S Yamanaka</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rais, Y" uniqKey="Rais Y">Y Rais</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Di Stefano, B" uniqKey="Di Stefano B">B Di Stefano</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hou, P" uniqKey="Hou P">P Hou</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ichida, Jk" uniqKey="Ichida J">JK Ichida</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Li, R" uniqKey="Li R">R Li</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Maherali, N" uniqKey="Maherali N">N Maherali</name></author><author><name sortKey="Hochedlinger, K" uniqKey="Hochedlinger K">K Hochedlinger</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mikkelsen, Ts" uniqKey="Mikkelsen T">TS Mikkelsen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Samavarchi Tehrani, P" uniqKey="Samavarchi Tehrani P">P Samavarchi-Tehrani</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Meissner, A" uniqKey="Meissner A">A Meissner</name></author><author><name sortKey="Wernig, M" uniqKey="Wernig M">M Wernig</name></author><author><name sortKey="Jaenisch, R" uniqKey="Jaenisch R">R Jaenisch</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bandura, Dr" uniqKey="Bandura D">DR Bandura</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Qiu, P" uniqKey="Qiu P">P Qiu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhang, J" uniqKey="Zhang J">J Zhang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Takahashi, K" uniqKey="Takahashi K">K Takahashi</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<pmc-dir>properties manuscript</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-journal-id">0410462</journal-id><journal-id journal-id-type="pubmed-jr-id">6011</journal-id><journal-id journal-id-type="nlm-ta">Nature</journal-id><journal-id journal-id-type="iso-abbrev">Nature</journal-id><journal-title-group><journal-title>Nature</journal-title></journal-title-group><issn pub-type="ppub">0028-0836</issn><issn pub-type="epub">1476-4687</issn></journal-meta><article-meta><article-id pub-id-type="pmid">25830878</article-id><article-id pub-id-type="pmc">4441548</article-id><article-id pub-id-type="doi">10.1038/nature14274</article-id><article-id pub-id-type="manuscript">NIHMS660783</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Early reprogramming regulators identified by prospective isolation and mass cytometry</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Lujan</surname><given-names>Ernesto</given-names></name><xref ref-type="aff" rid="A1">1</xref><xref ref-type="aff" rid="A2">2</xref><xref ref-type="aff" rid="A4">4</xref><xref ref-type="author-notes" rid="FN1">*</xref></contrib><contrib contrib-type="author"><name><surname>Zunder</surname><given-names>Eli R.</given-names></name><xref ref-type="aff" rid="A3">3</xref><xref ref-type="author-notes" rid="FN1">*</xref></contrib><contrib contrib-type="author"><name><surname>Ng</surname><given-names>Yi Han</given-names></name><xref ref-type="aff" rid="A1">1</xref><xref ref-type="aff" rid="A4">4</xref><xref ref-type="aff" rid="A5">5</xref></contrib><contrib contrib-type="author"><name><surname>Goronzy</surname><given-names>Isabel N.</given-names></name><xref ref-type="aff" rid="A1">1</xref><xref ref-type="aff" rid="A4">4</xref></contrib><contrib contrib-type="author"><name><surname>Nolan</surname><given-names>Garry P.</given-names></name><xref ref-type="aff" rid="A3">3</xref></contrib><contrib contrib-type="author"><name><surname>Wernig</surname><given-names>Marius</given-names></name><xref ref-type="aff" rid="A1">1</xref><xref ref-type="aff" rid="A4">4</xref></contrib></contrib-group><aff id="A1"><label>1</label>Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA 94305, USA</aff><aff id="A2"><label>2</label>Department of Genetics, Stanford University, Stanford, CA 94305, USA</aff><aff id="A3"><label>3</label>Baxter Laboratory in Stem Cell Biology, Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA</aff><aff id="A4"><label>4</label>Department of Pathology, Stanford University, Stanford, CA 94305, USA</aff><aff id="A5"><label>5</label>Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA</aff><author-notes><corresp id="cor1">Correspondence and requests for materials should be addressed to M.W. (<email>wernig@stanford.edu</email>)</corresp><fn id="FN1" fn-type="equal"><label>*</label><p id="P1">These authors contributed equally to this work.</p></fn></author-notes><pub-date pub-type="nihms-submitted"><day>5</day><month>2</month><year>2015</year></pub-date><pub-date pub-type="epub"><day>01</day><month>4</month><year>2015</year></pub-date><pub-date pub-type="ppub"><day>21</day><month>5</month><year>2015</year></pub-date><pub-date pub-type="pmc-release"><day>21</day><month>11</month><year>2015</year></pub-date><volume>521</volume><issue>7552</issue><fpage>352</fpage><lpage>356</lpage><pmc-comment>elocation-id from pubmed: 10.1038/nature14274</pmc-comment>
<permissions><license xlink:href="http://www.nature.com/authors/editorial_policies/license.html#terms"><license-p>Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:<ext-link ext-link-type="uri" xlink:href="http://www.nature.com/authors/editorial_policies/license.html#terms">http://www.nature.com/authors/editorial_policies/license.html#terms</ext-link></license-p></license></permissions><abstract><p id="P2">In the context of most induced pluripotent stem (iPS) cell reprogramming methods, heterogeneous populations of nonproductive and staggered productive intermediates arise at different reprogramming time points<sup><xref rid="R1" ref-type="bibr">1</xref>–<xref rid="R11" ref-type="bibr">11</xref></sup>. Despite recent reports claiming substantially increased reprogramming efficiencies using genetically modified donor cells<sup><xref rid="R12" ref-type="bibr">12</xref>,<xref rid="R13" ref-type="bibr">13</xref></sup> prospectively isolating distinct reprogramming intermediates remains an important goal to decipher reprogramming mechanisms. Previous attempts to identify surface markers of intermediate cell populations were based on the assumption that during reprogramming cells progressively lose donor cell identity and gradually acquire iPS cell properties<sup><xref rid="R1" ref-type="bibr">1</xref>,<xref rid="R2" ref-type="bibr">2</xref>,<xref rid="R7" ref-type="bibr">7</xref>,<xref rid="R8" ref-type="bibr">8</xref>,<xref rid="R10" ref-type="bibr">10</xref></sup>. Here, we report that iPS cell and epithelial markers, such as SSEA1 and EpCAM, respectively, are not predictive of reprogramming during early phases. Instead, in a systematic functional surface marker screen we find that early reprogramming-prone cells express a unique set of surface markers, including CD73, CD49d and CD200 that are absent in fibroblasts and iPS cells. Single cell mass cytometry and prospective isolation show that these distinct intermediates are transient and bridge the gap between donor cell silencing and pluripotency marker acquisition during the early, presumably stochastic reprogramming phase<sup><xref rid="R2" ref-type="bibr">2</xref></sup>. Expression profiling revealed early upregulation of the transcriptional regulators <italic>Nr0b1</italic> and <italic>Etv5</italic> in this reprogramming state, preceding activation of key pluripotency regulators such as <italic>Rex1, Dppa2, Nanog</italic> and <italic>Sox2</italic>. Both factors are required for the generation of the early intermediate state and fully reprogrammed iPS cells, and thus mark some of the earliest known regulators of iPS cell induction. Our study deconvolutes the first steps in a hierarchical series of events that lead to pluripotency acquisition.</p></abstract></article-meta></front><body><p id="P3">Reprogramming somatic cells to a pluripotent state by forced transcription factor expression is typically an inefficient process involving heterogeneous populations that impede molecular analysis of productive reprogramming<sup><xref rid="R1" ref-type="bibr">1</xref>–<xref rid="R11" ref-type="bibr">11</xref>,<xref rid="R14" ref-type="bibr">14</xref></sup>. Previous studies have shown that reprogramming is a multi-stage process involving a presumably early stochastic and late deterministic phase<sup><xref rid="R2" ref-type="bibr">2</xref>,<xref rid="R3" ref-type="bibr">3</xref>,<xref rid="R5" ref-type="bibr">5</xref></sup>. Progress has been made characterizing intermediates of the late phase given the appearance of well-known pluripotency markers at that time<sup><xref rid="R1" ref-type="bibr">1</xref>,<xref rid="R2" ref-type="bibr">2</xref>,<xref rid="R7" ref-type="bibr">7</xref>,<xref rid="R8" ref-type="bibr">8</xref>,<xref rid="R10" ref-type="bibr">10</xref></sup>. In contrast, not much is known about the early stochastic phase except the consistent observation that downregulation of donor cell markers is an early feature of successful reprogramming<sup><xref rid="R1" ref-type="bibr">1</xref>,<xref rid="R2" ref-type="bibr">2</xref>,<xref rid="R7" ref-type="bibr">7</xref>,<xref rid="R8" ref-type="bibr">8</xref>,<xref rid="R10" ref-type="bibr">10</xref>,<xref rid="R15" ref-type="bibr">15</xref>–<xref rid="R19" ref-type="bibr">19</xref></sup>.</p><p id="P4">To identify surface markers of early reprogramming stages, we screened 176 antibodies on cells representing three stages of the reprogramming process: 1) mouse embryonic fibroblasts (MEFs), 2) a previously characterized partially reprogrammed cell (PRC) line<sup><xref rid="R18" ref-type="bibr">18</xref>,<xref rid="R20" ref-type="bibr">20</xref></sup> and 3) embryonic stem (ES) cells. We identified 21 markers enriched or shared between these cell types and characterized their co-expression by single cell mass cytometry using spanning-tree progression analysis of density-normalized events (SPADE), which groups similar cells into a defined number of clusters<sup><xref rid="R21" ref-type="bibr">21</xref>,<xref rid="R22" ref-type="bibr">22</xref></sup>. (<xref ref-type="fig" rid="F1">Fig. 1a,b</xref> and <xref ref-type="fig" rid="F5">Extended Data Fig. 1</xref>). Next, we characterized their expression by mass cytometry during <italic>Oct4, Sox2, Klf4</italic> and <italic>c-Myc</italic>-driven MEF reprogramming. By day 3, downregulation of the fibroblast expression program was evident (<xref ref-type="fig" rid="F1">Fig. 1c</xref> and <xref ref-type="fig" rid="F6">Extended Data Fig. 2</xref> and <xref ref-type="fig" rid="F7">3</xref>). At day 6, major branches were delineated by the PRC marker CD73 and ESC markers CD54, CD326 and SSEA1. Little co-expression was observed between these markers suggesting several intermediates arise during early reprogramming or early expression of some of these markers may not be indicative of productive reprogramming. By day 9, CD326 and SSEA1 expression converged in a subpopulation and persisted on days 12 and 16 (<xref ref-type="fig" rid="F6">Extended Data Fig. 2b</xref>). These clusters were heterogeneous for CD73, suggesting they may be derivatives of separate populations or a CD73<sup>high</sup> subpopulation becomes CD326<sup>high</sup>, SSEA1<sup>high</sup>. Over our time course, the ESC marker CD54 largely localized to fibroblast branches and did not cluster with CD326<sup>high</sup> and SSEA1<sup>high</sup> clusters, suggesting CD54 expression in pluripotent cells is a late event<sup><xref rid="R7" ref-type="bibr">7</xref></sup>.</p><p id="P5">Hypothesizing that cells destined to successfully reprogram, acquire surface markers in a stepwise, non-stochastic manner during early reprogramming, we assessed reprogramming efficiencies for cells with high or low expression of the above-characterized surface markers at early time points (<xref ref-type="fig" rid="F2">Fig. 2a–c</xref> and <xref ref-type="fig" rid="F8">Extended Data Fig. 4a</xref>). As mass culture experiments can be misleading since a single proliferative population can seed multiple secondary colonies, we conducted 96-well assays to assess unique reprogramming events. Based on the current literature it would be expected that reprogramming cells would be enriched by (i) low levels of fibroblast markers and (ii) high levels of ESC markers independent of the time and state of reprogramming<sup><xref rid="R2" ref-type="bibr">2</xref>,<xref rid="R7" ref-type="bibr">7</xref>,<xref rid="R8" ref-type="bibr">8</xref>,<xref rid="R10" ref-type="bibr">10</xref></sup>. Indeed, by day 3, populations expressing low levels of all fibroblast markers except CD47 enriched for reprogramming populations when compared to highly expressing populations (<xref ref-type="fig" rid="F2">Fig. 2a</xref>). Surprisingly though, at these early timepoints, cells with high levels of the ESC markers SSEA1, CD54, CD326, or CD71 did not show significantly increased reprogramming (<xref ref-type="fig" rid="F2">Fig. 2a–c</xref>). Day 9 fractions expressing high levels of CD326 or SSEA1 began to show greater but insignificant enrichment for reprogramming populations. We confirmed previous reports that SSEA1-sorted cells produce more iPS cell colonies in mass culture<sup><xref rid="R1" ref-type="bibr">1</xref>,<xref rid="R8" ref-type="bibr">8</xref>,<xref rid="R10" ref-type="bibr">10</xref></sup> emphasizing the critical importance of the 96-well assay (<xref ref-type="fig" rid="F8">Extended Data Fig. 4b,c</xref>). Unlike previously assumed, our findings demonstrate that acquisition of markers that define the pluripotent state is a late event and early expression of ESC markers has little predictive value for successful reprogramming. Additionally these data support that the mesenchymal-to-epithelial transition (MET) as judged by the epithelial marker CD326 (EpCAM) is a late event<sup><xref rid="R2" ref-type="bibr">2</xref>,<xref rid="R15" ref-type="bibr">15</xref>–<xref rid="R17" ref-type="bibr">17</xref>,<xref rid="R19" ref-type="bibr">19</xref></sup>.</p><p id="P6">Our surface marker screen identified several specific markers for stable partially reprogrammed cells<sup><xref rid="R4" ref-type="bibr">4</xref>,<xref rid="R9" ref-type="bibr">9</xref>,<xref rid="R18" ref-type="bibr">18</xref>,<xref rid="R20" ref-type="bibr">20</xref></sup>. Though thought to be ‘stuck’ during reprogramming<sup><xref rid="R4" ref-type="bibr">4</xref>,<xref rid="R18" ref-type="bibr">18</xref>,<xref rid="R20" ref-type="bibr">20</xref></sup>, we hypothesized a productive intermediate might arise between fibroblasts and iPS cells that share a subset of these markers. Indeed, day 6 fractions expressing high levels of the PRC markers CD73, CD49d and CD200 significantly enriched for reprogramming populations (<xref ref-type="fig" rid="F2">Fig. 2b</xref>). Also on day 9 the CD73<sup>high</sup> and CD49d<sup>high</sup> populations contained higher reprogramming activity (<xref ref-type="fig" rid="F2">Fig. 2c</xref>). In agreement with these results, the day 6 SPADE analysis showed CD73<sup>high</sup>, CD49d<sup>high</sup> and CD200<sup>high</sup> branches largely clustering independently from branches enriched for ESC and MEF markers (<xref ref-type="fig" rid="F1">Fig. 1c</xref>). These results demonstrate that distinct intermediate populations arise after fibroblast program repression but before ESC marker acquisition.</p><p id="P7">We next focused on the markers CD73 and CD49d. When corrected for plating efficiency both CD73<sup>high</sup> and CD49d<sup>high</sup> populations showed remarkably high reprogramming efficiencies of 9.5% +/− 3.5 and 12.5% +/− 5.7, respectively (<xref ref-type="fig" rid="F2">Fig. 2d–f</xref>). Similar enrichment of a reprogramming-prone population was observed in reprogramming tail tip fibroblasts (TTFs) and glial-restricted neural precursor cells suggesting CD73 and CD49d may be universal markers of intermediate reprogramming stages (<xref ref-type="fig" rid="F3">Fig. 3a–d</xref>). Finally, we explored their potential functional implications during reprogramming, and observed that adenosine, the enzymatic product of CD73, has a negative effect throughout and CD49d activity is necessary during late reprogramming (<xref ref-type="fig" rid="F8">Extended Data Fig. 4e</xref>).</p><p id="P8">We then used our day 6 SPADE analysis to identify heterogeneously expressed markers that could subdivide the CD73<sup>high</sup> reprogramming-prone population and conducted single cell efficiency assays (<xref ref-type="fig" rid="F3">Fig. 3e,f</xref>). Within the CD73-positive population the CD44<sup>high</sup>, CD71<sup>low</sup> and CD326<sup>high</sup> fractions failed to reprogram, while CD49d<sup>high</sup> and CD326<sup>low</sup> fractions enriched for a reprogramming population. Thus, a CD73<sup>high</sup> CD49d<sup>high</sup> CD326<sup>low</sup> CD44<sup>low</sup> signature best describes the population undergoing productive reprogramming on day 6. Overlaying this signature onto the day 6 SPADE tree allowed for determination of the exact cellular clusters most similar to this reprogramming-prone signature (<xref ref-type="fig" rid="F3">Fig. 3g</xref>). As expected, MEF markers were low in these poised populations. This suggests this intermediate arises after loss of mesenchymal markers, but before MET completion as indicated by reprogramming enrichment in the CD326<sup>low</sup> fraction.</p><p id="P9">To identify subsequent reprogramming stages we conducted continuation analysis where cells were sorted on day 6 and characterized by mass cytometry on day 16 (<xref ref-type="fig" rid="F3">Fig. 3h</xref>, <xref ref-type="fig" rid="F8">Extended Data Fig. 4</xref>–<xref ref-type="fig" rid="F10">6</xref>). By day 10, reprogramming-prone populations formed distinct colonies with ESC-like morphology, while CD73<sup>low</sup> cells were highly proliferative but failed to develop into mature colonies (<xref ref-type="fig" rid="F9">Extended Data Fig. 5b</xref>). Continuation analysis on day 16 revealed that while reprogramming-prone and non-prone populations contained CD326 expressing cells, broad overlap between CD326<sup>high</sup> and SSEA1<sup>high</sup> clusters was only in mature reprogramming-prone populations (<xref ref-type="fig" rid="F3">Fig. 3h</xref>, <xref ref-type="fig" rid="F10">Extended Data Fig. 6</xref>). These clusters did not overlap with the ESC marker CD54, and were heterogeneous for CD73 and CD49d. We conclude a distinct CD326<sup>high</sup>, SSEA1<sup>high</sup>, CD54<sup>low</sup> intermediate arises after the CD73<sup>high</sup>/CD49d<sup>high</sup> intermediate and before pluripotency acquisition.</p><p id="P10">We then used the intermediates stages to gain molecular insights into transcriptional regulation of early reprogramming. Gene expression analysis intriguingly showed these intermediates precede activation of the majority of transcription factors thought to be predictive markers for pluripotency induction (<xref ref-type="fig" rid="F4">Fig. 4a</xref> and <xref ref-type="fig" rid="F11">Extended Data Fig. 7</xref>)<sup><xref rid="R2" ref-type="bibr">2</xref>,<xref rid="R7" ref-type="bibr">7</xref></sup>. The observation that these intermediates arise prior to key pluripotency regulators suggests a separate combination of early transcription factors must be induced to generate early intermediates and poise them for pluripotency acquisition. We found the transcription factors <italic>Nr0b1</italic> and <italic>Etv5</italic> preferentially expressed in reprogramming-prone populations and highly expressed in ESCs suggesting a functional role in poising early reprogramming (<xref ref-type="fig" rid="F4">Fig. 4a</xref>, <xref ref-type="fig" rid="F11">Extended Data Fig. 7b–d</xref>).</p><p id="P11">To assess whether these genes were necessary to induce the early intermediate populations we generated three short hairpins against each gene (<xref ref-type="fig" rid="F11">Extended Data Fig. 7e,f</xref>). We then assessed the ability of reprogramming MEFs infected with these short hairpins to induce CD73<sup>high</sup>/CD49d<sup>high</sup> intermediates 9 days after reprogramming induction (<xref ref-type="fig" rid="F4">Fig. 4b</xref> and <xref ref-type="fig" rid="F11">Extended Data Fig. 7g</xref>). Surprisingly, while MEFs infected with a control short hairpin were able to induce the CD73<sup>high</sup>/CD49<sup>high</sup> intermediate (6.70 +/− 2.27%), reprogramming MEFs infected with short hairpins targeting <italic>Etv5</italic> or <italic>Nr0b1</italic> were significantly impaired (<xref ref-type="fig" rid="F4">Fig. 4b</xref>). This phenotype could be rescued by cDNA overexpression in combination with a hairpin targeting the UTR for the gene of interest (<xref ref-type="fig" rid="F4">Fig. 4c</xref>). Further, when <italic>Nanog</italic><sup>+</sup> colonies or <italic>Sox2-EGFP</italic><sup>+</sup> colonies were assessed 24 days after reprogramming induction, a dramatic decrease in reprogramming efficiencies was observed (<xref ref-type="fig" rid="F4">Fig. 4d,e</xref>). In contrast, knock-down of either gene in ESCs did not affect survival or proliferation (<xref ref-type="fig" rid="F11">Extended Data Fig. 7h</xref>). While this paper was under review an independent report confirmed <italic>Nr0b1</italic> as necessary for reprogramming<sup><xref rid="R23" ref-type="bibr">23</xref></sup>. These data indicate that <italic>Etv5</italic> and <italic>Nr0b1</italic> are required for the generation of the CD73<sup>high</sup>/CD49d<sup>high</sup> poised intermediate necessary to induce the canonical pluripotency program and definitive iPS cell formation.</p><p id="P12">We then wondered whether a similar intermediate population arises in high efficiency reprogramming systems<sup><xref rid="R12" ref-type="bibr">12</xref>,<xref rid="R13" ref-type="bibr">13</xref></sup>. Published expression analysis of two high efficiency systems showed transient CD73 upregulation, suggesting the presence of a similar intermediate (<xref ref-type="fig" rid="F12">Extended Data Fig. 8a,b</xref>). We then characterized one of these systems, the <italic>Mbd3<sup>fl/−</sup></italic> secondary MEF system, in greater detail<sup><xref rid="R12" ref-type="bibr">12</xref></sup>. After confirming reported reprogramming efficiencies, we analyzed this system by mass spectrometry (<xref ref-type="fig" rid="F12">Extended Data Figs. 8</xref>–<xref ref-type="fig" rid="F14">10</xref>). By day 3, fibroblast marker repression was evident, and CD73 was upregulated within this population (<xref ref-type="fig" rid="F12">Extended Data Fig. 8e</xref> and <xref ref-type="fig" rid="F13">9b</xref>). Within the CD73<sup>high</sup>/MEFSK4<sup>low</sup> population, CD49d (<italic>itga4</italic>) upregulation was not apparent, but we noticed the emergence of a separate integrin, CD104 (<italic>itgb4</italic>). By day 4, the major CD73<sup>high</sup> branch clearly overlapped with the CD104<sup>high</sup> branch and persisted into day 5. SSEA1<sup>high</sup> and CD326<sup>high</sup> expression was present on day 4, but clear coexpression was not seen until day 5. By day 9, CD73 and CD104 expression was dramatically reduced while CD326 and SSEA1 expression remained high. These data demonstrate a transient CD73<sup>high</sup>/CD104<sup>high</sup> population arises after donor cell program repression and prior to ESC marker acquisition even in a highly efficient reprogramming system. Similar to CD49d and CD73, CD104 is not highly expressed in ESCs (<xref ref-type="fig" rid="F1">Fig. 1a</xref>). Similar to viral reprogramming, adenosine treatment abolished reprogramming in the Mbd3 reprogramming system, albeit only at late stages, whereas compounds affecting CD49d function had little effect (<xref ref-type="fig" rid="F8">Extended Data Fig. 4f</xref>).</p><p id="P13">The stage-specific framework provided in this study bridges the previously unexplored gap between donor program silencing and pluripotent marker acquisition (<xref ref-type="fig" rid="F4">Fig. 4f</xref>). We demonstrate a transient, ‘poised’ intermediate present across multiple reprogramming systems suggesting a general property of iPSC reprogramming. We note similar to SSEA1, TRA-1-60 enriches for reprogramming-prone intermediates at later time points during human reprogramming<sup><xref rid="R11" ref-type="bibr">11</xref>,<xref rid="R24" ref-type="bibr">24</xref></sup>, and we speculate a similar transient intermediate arises in the human system.</p><sec sec-type="methods" id="S1"><title>Methods</title><sec id="S2"><title>Cell Culture</title><p id="P14">Embryonic fibroblasts were isolated from E13.5 embryos derived from B6;129S-<italic>Sox2<sup>EGFP/+</sup></italic> mated with B6.Cg-<italic>Gt(ROSA)26Sor<sup>tm1(rtTA*M2)Jae</sup></italic> (allele referred to as <italic>Rosa-rtTA</italic>) as previously described<sup><xref rid="R25" ref-type="bibr">25</xref>,<xref rid="R26" ref-type="bibr">26</xref></sup>. Tail tip fibroblasts were derived from 1 week old B6 mice. This was done by chilling the animals on ice for 5 minutes, cutting the tail, and mincing in a 6cm dish in 1ml 0.25% trypsin. Another 1ml of 0.25% trypsin was added after mincing and this was incubated at 37C for 10 minutes. The mixture was then resuspended in 15ml MEF medium (described below) and plated on a 0.2% gelatinized 15cm plate. Glial cultures were prepared from CD1 mice as previously described<sup><xref rid="R25" ref-type="bibr">25</xref></sup>. MEFs, TTFs and glial cells were cultured in MEF medium which consisted of 10% cosmic calf serum (Thermo Scientific) in DMEM (Invitrogen) supplemented with non-essential amino acids (Invitrogen), penicillin-streptomycin (Invitrogen), sodium pyruvate (Invitrogen) and 2-mercaptoethanol (Invitrogen). Primary <italic>Mbd3<sup>fl/−</sup>, Oct4-GFP</italic> secondary MEFs<sup><xref rid="R12" ref-type="bibr">12</xref></sup> were a generous gift from Jacob Hanna and were grown in MEF medium. We verified modification of the <italic>Mbd3</italic> locus by Western blot analysis (described below under ‘Western blotting’, <xref ref-type="fig" rid="F12">Extended Data Figure 8f</xref>). Our partially reprogrammed line was previously characterized<sup><xref rid="R20" ref-type="bibr">20</xref></sup>. These were grown in MEF medium supplemented with 15% cosmic calf serum. <italic>Oct4-neoR</italic> knock-in mouse ES cells<sup><xref rid="R18" ref-type="bibr">18</xref>,<xref rid="R20" ref-type="bibr">20</xref></sup> were grown in mESC medium which consisted of 12% knockout replacement serum (Invitrogen), 3% cosmic calf serum and supplemented with non-essential amino acids, penicillin-streptomycin, sodium pyruvate, 2-mercaptoethanol and LIF. MEFs, PRCs and mESCs were routinely tested for mycoplasma contamination.</p></sec><sec id="S3"><title>Surface marker screening</title><p id="P15">We screened mouse surface markers using the “Mouse cell surface marker screening panel” Lyoplate (BD Biosciences, material number 562208) according to the manufacturer’s instructions. In brief, 150×10^6 cells were used for our partially reprogrammed cells (passage 15) and <italic>Oct4-neoR</italic> mES cells. Cells were plated on 0.2% gelatin coated plates and treated with neomycin three days before screening. 175×10^6 cells were used for passage 4 <italic>Sox2-EGFP</italic> MEFs. Cells were washed with PBS-EDTA, dissociated in 10× TrypLE (Invitrogen) for 5 minutes, washed once in PBS before staining for 30 minutes in primary antibody in staining medium (PBS-EDTA supplemented with 0.5% BSA) according to manufacturer’s instructions, washing once in PBS, staining for 30 minutes in fluorophore conjugated secondary antibody in staining medium, washing once in PBS and analyzed on a BD LSR Fortessa FACS analyzer in in staining medium. We used a high concentration of TrypLE to verify that all identified markers would not be cleaved by our dissociation reagent. We further validated a subset of these identified makers with our control populations dissociated in 1× TrypLE and stained with fluorophore conjugated antibodies as described below.</p></sec><sec id="S4"><title>Reprogramming</title><p id="P16">Reprogramming assays were conducted with passage 4 <italic>Rosa-rtTA<sup>+/−</sup>; Sox2<sup>EGFP/+</sup></italic> or <italic>Rosa-rtTA<sup>+/−</sup></italic> (derived from the same litter – see mating scheme above) mouse embryonic fibroblasts, passage 2 B6 tail tip fibroblasts or passage 3 B6 glia as indicated. FUW-tetO lentiviral vectors and lentiviral packaging were used as previously described<sup><xref rid="R25" ref-type="bibr">25</xref></sup>. Passage 3 MEFs and passage 2 glial cells were split onto 10cm 0.2% gelatin coated plates at 250,000 cells per plate one day before infection. Passage 1 TTFs were split onto 10cm 0.2% gelatin coated plates at 100,000 cells per plate one day before infection. Cells were infected in MEF medium supplemented with polybrene (8 µg/mL; Sigma). One day after infection, MEF medium supplemented with doxycycline was added and this was considered to be day 0. Media was replaced every 2 days. On day 16, medium was replaced with mES cell medium without doxycycline. Due to the complication of getting a large number of glial cells, we performed the reprogramming efficiency assays once per time point (day 6 and 9) but both were done with independently derived primary cells from different animals, independently derived virus and sorts.</p><p id="P17">The following compounds were also added to reprogramming medium on the days indicated in the text: BIO 1211 (Tocris, 4nM), adenosine (Sigma, 20µM or 2 µM as indicated), fibronectin (Sigma, 4µg/ml), AMP-CP [Adenosine 5′-(α,β-methylene)diphosphate] (Sigma, 20µM). These were chosen as CD73 (<italic>5’-nucleotidase, ecto</italic>) converts AMP to adenosine, CD49d (<italic>integrin α4</italic>) is an integrin whose substrates are VCAM1 and fibronectin, α,β-Methyleneadenosine 5′-diphosphate (AMP-CP) is a CD73 inhibitor and BIO1211 is CD49d inhibitor</p><p id="P18">Hairpins for <italic>Nr0b1</italic> and <italic>Etv5</italic> were designed with the pSico oligomaker (<xref ref-type="supplementary-material" rid="SD1">Supplementary Table 6</xref>) and cloned into the lentiviral pSico-puro vector<sup><xref rid="R27" ref-type="bibr">27</xref></sup> (<ext-link ext-link-type="uri" xlink:href="http://web.mit.edu/jacks-lab/protocols/pSico.html">http://web.mit.edu/jacks-lab/protocols/pSico.html</ext-link>). We assessed knockdown efficiencies in partially reprogrammed cells as they express these genes and grow well in MEF medium supplemented with 15% serum alone. To assess knockdown efficiencies, 50,000 PRCs were plated onto a 6 well gelatin coated well one day before transduction. Cells were transduced with the hairpin of interest in MEF medium supplemented with 15% serum and polybrene (8 µg/mL). The medium was exchanged the following day with MEF medium supplemented with 15% serum and puromycin (2µg/ml). Cells were cultured for a further three days before RNA extraction (for a total of four days after infection). RNA was prepared with an RNeasy purification kit as described below.</p><p id="P19">To assess the effects of knockdown of these genes on the day 9 CD73<sup>high</sup>/CD49d<sup>high</sup> intermediate, 100,000 P4 <italic>Rosa-rtTA</italic> MEFs were plated on a 10cm gelatin coated plate. These were transduced the following day with the doxycycline-inducible FUW-tetO-<italic>Klf4, Oct-3/4, Sox2</italic> and <italic>c-Myc</italic> vectors and indicated hairpins as described above. Medium was supplemented with doxycycline one day after infection (day 0). We note that because <italic>Rosa-rtTA</italic> MEFs contain a PGK-puromycin cassette, they were not selected with puromycin. On day 9, CD73-Alexa 488 (1:50) and CD49d-Alexa 750 (1:50) were analyzed by FACS (FACS staining described below). To assess reprogramming efficiencies for these genes, 30,000 P4 <italic>Rosa-rtTA</italic> MEFs were plated on a 6cm gelatin coated plate or 15,000 P4 <italic>Rosa-rtTA, Sox2-EGFP</italic> MEFs were plated on a 6 well gelatin coated well and transduced the following day in the same manner. At day 16, the culture medium was switched to mES cell medium without doxycycline. 24 days after reprogramming induction, <italic>Nanog</italic><sup>+</sup> colonies were assessed for <italic>Rosa-rtTA</italic> MEFs (immunofluorescence staining described below) or <italic>Sox2-EGFP</italic><sup>+</sup> colonies for <italic>Rosa-rtTA, Sox2-EGFP</italic> MEFs. In total, three independent reprogramming efficient experiments were conducted across the two different MEF lines. To further validate the specificity of the effects, we have performed a "rescue" experiment to demonstrate that upon re-expression of the cDNA (under knock-down conditions) the effect is eliminated. To this end, the cDNAs for <italic>Nr0b1</italic> and <italic>Etv5</italic> were cloned into the FUW-tetO vector. 15,000 P4 <italic>Rosa-rtTA</italic> MEFs on 6 well gelatin coated plates were then transduced the doxycycline-inducible FUW-tetO-<italic>Klf4, Oct-3/4, Sox2</italic> and <italic>c-Myc</italic> vectors and indicated hairpins with the FUW-tetO-cDNA (<italic>Etv5</italic> or <italic>Nr0b1</italic>) or an empty vector. Medium was supplemented with doxycycline one day after infection (day 0). On day 9, CD73-Alexa 488 (1:50) and CD49d-Alexa 750 (1:50) were analyzed by FACS (FACS staining described below). As the rescue experiment was supplementary to and consistent with the main knockdown experiments, we conducted only one rescue experiment for one hairpin.</p><p id="P20">To verify that mouse ESCs could survive and proliferate after knock-down of <italic>Etv5</italic> or <italic>Nr0b1</italic>, we infected 30,000 ESCs per well in gelatinized 6-well plates in mESC medium and replaced the medium the following day with mESC medium supplemented with puromycin to select for successful transduction. These were then cultured for 3 days, dissociated with 0.25% trypsin and replated onto gelatinized 6 well plates. These were then cultured for 3 days, fixed and stained for <italic>Oct4</italic> (described below).</p><p id="P21">To reprogram <italic>Mbd3<sup>fl/−</sup>, Oct4-GFP</italic> secondary MEFs (the <italic>Oct4-GFP</italic> allele in these cells is not a targeted, but well-characterized, transgenic reporter<sup><xref rid="R12" ref-type="bibr">12</xref></sup>), we first assayed reprogramming conditions with 2000 <italic>Mbd3<sup>fl/−</sup></italic> secondary MEFs on 2×10<sup>6</sup> mitomycin (Sigma) treated B6 feeders, 10<sup>5</sup><italic>Mbd3<sup>fl/−</sup></italic> secondary MEFs without feeders and 2×10<sup>5</sup><italic>Mbd3<sup>fl/−</sup></italic> secondary MEFs without feeders on 10cm dishes coated with 0.2% gelatin. We found optimal reprogramming efficiencies with 2000 <italic>Mbd3<sup>fl/−</sup></italic> secondary MEFs and 2×10<sup>6</sup> feeders as previously reported<sup><xref rid="R12" ref-type="bibr">12</xref></sup>. All reprogramming assays were done in non-hypoxic conditions. To reprogram cells, cells were cultured in media 1 [recombinant human LIF (10ng/ml, Peprotech), doxycycline (1µg/ml) and ascorbic acid (10µg/ml, Sigma) in 15% cosmic calf serum (Thermo Scientific) in DMEM (Invitrogen) supplemented with non-essential amino acids (Invitrogen), penicillin-streptomycin (Invitrogen), sodium pyruvate (Invitrogen) and 2-mercaptoethanol (Invitrogen)] for 3 days and then media 2 [recombinant human LIF (10ng/ml), doxycycline (1µg/ml), PD0325901 (1µM, Cell Signaling) and CHIR99021 (3µM, Cayman) in 15% knockout replacement serum (Invitrogen) in DMEM (Invitrogen) supplemented with non-essential amino acids (Invitrogen), penicillin-streptomycin (Invitrogen), sodium pyruvate (Invitrogen) and 2-mercaptoethanol (Invitrogen),] until the completion of the experiment. Media was exchanged every 2 days.</p></sec><sec id="S5"><title>Mass Cytometry Analysis</title><p id="P22">On designated days the reprogramming cultures were treated with 1× TrypLE (Invitrogen) for 5 minutes at 37C, dissociated into single cell suspension by trituration, and then washed twice with PBS. The cell samples were then incubated with metal-conjugated antibodies (<xref ref-type="supplementary-material" rid="SD1">Supplementary Table 1</xref>) in PBS containing 5% FBS (Omega Scientific) for 30 minutes on ice, washed once with PBS containing 5% FBS, treated with 25 µM cisplatin for 1 minute on ice for live-dead cell discrimination, washed once with PBS containing 5% FBS, and then fixed with 1.6% paraformaldehyde at room temperature for 10 minutes. Formaldehyde-fixed cell samples were then permeabilized with methanol on ice for 15 minutes, washed one time with PBS containing 0.5% BSA, and then incubated at room temperature for 15 minutes with an Irridium-containing DNA intercalator (DVS Sciences/Fluidigm) in PBS containing 1.6% paraformaldehyde. After intercalation/fixation, the cell samples were washed once with PBS containing 0.5% BSA and twice with water before measurement on a CyTOF mass cytometer (DVS Sciences/Fluidigm). Normalization for detector sensitivity was performed as previously described<sup><xref rid="R28" ref-type="bibr">28</xref></sup>, using polystyrene normalization beads containing Lanthanum-139, Praseodymium-141, Terbium-159, Thulium-169, and Lutetium-175.</p></sec><sec id="S6"><title>SPADE Analysis</title><p id="P23">Density-dependent downsampling, hierarchical clustering, cluster upsampling, and extraction of parameter medians was performed by the SPADE package (<ext-link ext-link-type="uri" xlink:href="http://www.cytospade.org">www.cytospade.org</ext-link>) as described in the main text and as previously described<sup><xref rid="R22" ref-type="bibr">22</xref>,<xref rid="R29" ref-type="bibr">29</xref></sup>. All assayed surface markers were used in the clustering step unless otherwise indicated, and the parameters for downsampling percentile and target number of clusters were set to 5% and 500, respectively.</p><p id="P24">The refined poised signature shown in <xref ref-type="fig" rid="F3">figure 3g</xref> was determined by calculating the similarity of each SPADE cluster to the hand-gated CD73<sup>high</sup> CD49d<sup>high</sup> CD44<sup>low</sup> CD326<sup>low</sup> population. Similarity was calculated by the Manhattan distance metric using all measured surface markers, and is indicated by the color scale bar (low distance equals high similarity).</p></sec><sec id="S7"><title>Immunofluorescence</title><p id="P25">Plates were fixed in 4% PFA for 10 minutes, washed 3 times with PBS, blocked and permeabilized in PBS supplemented with 5% CCS and 0.1% Triton-X 100 (Sigma) (blocking solution) for 10 minutes. 96 well plates were then incubated with mouse anti-Nanog (1:500, BD) or mouse anti-Oct4 (1:200, Santa Cruz Biotechnology) in blocking solution for 30 minutes, washed 3 times with PBS, incubated with donkey anti-mouse Alexa-555 (1:1000, Invitrogen) or anti-mouse Alexa-488 (1:1000, Invitrogen) in blocking solution for 30 minutes, washed three times with PBS and stained with DAPI for three minutes. Cells were then washed with PBS and visualized.</p></sec><sec id="S8"><title>Fluorescent activated cell sorting and efficiency assays</title><p id="P26">Cells were washed with PBS-EDTA, dissociated in 1× TrypLE for 5 minutes, washed with PBS, and incubated on ice with a fluorophore conjugated antibody and DAPI for 30 minutes in PBS-EDTA supplemented with 0.5% BSA. The sources and detailed descriptions of all antibodies used are listed in <xref ref-type="supplementary-material" rid="SD1">Supplementary Table 2</xref>. For 96 well assays, single or 20 DAPI<sup>−</sup> cells per well were double sorted on the indicated day into gelatinized 96 well plates supplemented with 400,000 feeders per plate in MEF medium supplemented with doxycycline. For the primary sort, cells were sorted into PBS supplemented with 0.5% BSA, and for the secondary sort, cells were sorted directly into 96 well plates. Efficiency assays were conducted 24 days after transgene induction and determined by the number of wells with <italic>Sox2-EGFP</italic><sup>+</sup> colonies. For mass culture reprogramming, 10,000 SSEA1<sup>high</sup> or SSEA1<sup>low</sup> cells were double sorted 6 days after transgene induction onto 3cm gelatinized plates supplemented with feeders and <italic>Sox2-EGFP</italic><sup>+</sup> colonies were assayed 24 days after transgene induction. We note that we used the same SSEA1 clone and vendor as previously used for determining reprogramming efficiencies<sup><xref rid="R8" ref-type="bibr">8</xref>,<xref rid="R10" ref-type="bibr">10</xref></sup>. TTF and glial reprogramming efficiencies were determined by double sorting on the indicated days into 96 wells (as described above) and assaying for <italic>Nanog</italic> by immunofluorescence. Plating efficiencies were determined by infecting <italic>Sox2-EGFP</italic> MEFs with FUW-tetO-<italic>hygroB-T2A-EGFP</italic>, selecting for 5 days in hygromycin, and counting the number of wells with GFP<sup>+</sup> cells 24 hours after sorting. We note that for all assays where CD73 was used for sorting, CD73-Alexa 647 was used (gating shown in <xref ref-type="fig" rid="F8">Extended Data Fig. 4</xref>). For CD73xCD49d analysis, CD73-488 was used (<xref ref-type="fig" rid="F11">Extended Data Fig. 7g</xref>).</p></sec><sec id="S9"><title>RNA Preparation and Expression Analysis</title><p id="P27">RNA of reprogramming populations for microarray analysis was prepared from <italic>Rosa-rtTA<sup>+/−</sup></italic> day 6 and day 9 reprogramming cultures double sorted for CD73 or CD49d (as described above). RNA of control populations for microarray analysis was prepared from <italic>Rosa-rtTA<sup>+/−</sup></italic> MEFs (passage 4), partially reprogrammed cells (passage 10) and V6.5 mouse ESCs (passage 11). RNA was prepared with RNeasy Mini Kit (Qiagen) and DNA was removed by on-column RNase-Free DNase treatment (Qiagen) according to the manufacturer’s instructions. Mouse Gene 2.0 ST Arrays (Affymetrix) were prepared by the Stanford Protein and Nucleic Acid (PAN) Facility. Data was normalized and gene names were assigned by Partek Genomic Suite. For all analysis, non-coding transcripts were removed. Preprocessing (floor = 100, ceiling = 20000, min fold change = 2), K-means clustering (k=5, seed value = 12345) and hierarchical clustering of k-means clusters (Pearson correlation, pairwise complete-linkage), and heat maps were generated by Gene Pattern (<ext-link ext-link-type="uri" xlink:href="http://www.broadinstitute.org/cancer/software/genepattern/">http://www.broadinstitute.org/cancer/software/genepattern/</ext-link>). Microarray data can be accessed with accession number GSE62957 from the National Center for Biotechnology Information database.</p><p id="P28">For <xref ref-type="fig" rid="F4">figure 4a</xref>, genes were selected based on pluripotency associated genes characterized in previous studies<sup><xref rid="R2" ref-type="bibr">2</xref>,<xref rid="R7" ref-type="bibr">7</xref></sup> or from differential expression of sorted populations and ESCs. <italic>Oct4</italic> is not shown as the probe failed to detect expression in mESCs.</p><p id="P29">For qPCR analysis, cDNA was generated with SuperScript First-Strand Synthesis System (Invitrogen). Data was generated with the 7900HT Real-Time PCR System (Applied Biosystems). 6µL reactions were prepared with SYBR Green Real-Time PCR Master Mix (Life Technologies) under the following conditions: 50 °C for 2 min, 95 °C for 10 min, and 40 cycles of 15 s at 95 °C and 1 min at 60 °C. All expression was normalized to GAPDH before comparing to control hairpin expression levels. Refer to <xref ref-type="supplementary-material" rid="SD1">Supplementary Table 6</xref> for primer sequences.</p></sec><sec id="S10"><title>Western blotting</title><p id="P30">Passage 3 <italic>Rosa-rtTA<sup>+/−</sup></italic> MEFs or secondary <italic>Mbd3<sup>fl/−</sup>, Rosa26-CreER</italic> MEFs were grown in 10-cm tissue culture plates. Secondary <italic>Mbd3<sup>fl/−</sup></italic> MEFs were treated with 1µM 4OH-tamoxifen for 24 hours, then samples were cultured for a further 48 hours and dissociated with 0.25% Trypsin. The cell pellet was then lysed with Cell Lysis Buffer (200mM NaCL, 50mM Tris pH8.0, 1% Trion X-100, 5% glycerol). 20ug of soluble protein was run on a 4–12% gradient Bis-Tris gel (Life Technologies) and blotted onto PVDF membrane. After blocking, membrane was incubated with primary antibody against Mbd3 (1:1000, Bethyl Laboratories, A302-528A) for an hour at RT, washed 3 times with PBS with 0.1% Tween-20, and incubated with secondary antibody anti-rabbit HRP (1: 5000, Jackson Immuno, A5441) for an hour at RT.</p></sec></sec><sec sec-type="extended-data" id="S11"><title>Extended Data</title><fig id="F5" orientation="portrait" position="anchor"><label>Extended Data Figure 1</label><caption><title>Results from surface marker screen</title><p id="P31"><bold>a</bold>, Shown are surface markers detected in mouse embryonic fibroblasts (MEF), partially reprogrammed cells (PR) or mouse embryonic stem cells (ESC). Numbers indicate the percentage of each population positive for the marker of interest, relative to isotype control samples. Percentages are colored from green to red to signify high to low values. Markers are grouped for enrichment in single populations or shared between multiple populations. <bold>b</bold>, SPADE analysis for MEFs, mESCs and PRCs for surface markers analyzed (continued from <xref ref-type="fig" rid="F1">Fig. 1b</xref>). Colors bars (bottom) represent ArcSinh transformed counts for each marker.</p></caption><graphic xlink:href="nihms660783f5"></graphic></fig><fig id="F6" orientation="portrait" position="anchor"><label>Extended Data Figure 2</label><caption><title>SPADE and biaxial analysis for MEF reprogramming</title><p id="P32"><bold>a</bold>, SPADE analysis of lentiviral infected MEF reprogramming populations (continued from <xref ref-type="fig" rid="F1">Fig. 1c</xref>). Colors bars (bottom) represent ArcSinh-transformed counts for each marker. <bold>b</bold>, Biaxial plots for selected markers in control populations and during MEF reprogramming.</p></caption><graphic xlink:href="nihms660783f6"></graphic></fig><fig id="F7" orientation="portrait" position="anchor"><label>Extended Data Figure 3</label><caption><title>SPADE analysis for MEF reprogramming</title><p id="P33"><bold>a</bold>, SPADE analysis of lentiviral infected MEF reprogramming populations (continued from <xref ref-type="fig" rid="F1">Fig. 1c</xref>). Colors bars (bottom) represent ArcSinh-transformed counts for each marker. <bold>b</bold>, Day 12 and 16 time points for markers shown in <xref ref-type="fig" rid="F1">Fig. 1c</xref>). Color bar for % total represents absolute percentages.</p></caption><graphic xlink:href="nihms660783f7"></graphic></fig><fig id="F8" orientation="portrait" position="anchor"><label>Extended Data Figure 4</label><caption><title>Details for sorting experiments and chemical treatment assay</title><p id="P34"><bold>a</bold>, 96 well reprogramming assay. 20 cells/well sorted at days 3, 6 and 9. <italic>Sox2-EGFP</italic><sup>+</sup> colonies were assayed on day 24. <bold>b</bold>, Gating strategy for SSEA1 in controls and day 6 reprogramming population. High and low-expressing populations were determined based on MEF and ESC control levels. <bold>c</bold>, 10,000 SSEA1<sup>high</sup> (black bar) or SSEA1<sup>low</sup> (white bar) were sorted onto 3cm gelatinized plates with feeders. <italic>Sox2-EGFP</italic><sup>+</sup> colonies were counted on day 24. <bold>d</bold>, Gating strategy for CD73 and CD49d in controls and day 6 reprogramming population. High and low-expressing populations were determined based on MEF, PRC and ESC control levels. <bold>e–f</bold>, Treatment of reprogramming populations with compounds affecting CD73 and CD49d. Shown are 96 well reprogramming efficiencies for infected Rosa-rtTA Sox2-EGFP MEFs (e) or secondary Mbd3<sup>fl/−</sup> MEFs (f). Y-axis displays wells with Sox2-EGFP<sup>+</sup> colonies 24 days after infection (e) or wells with Oct4-EGFP<sup>+</sup> colonies 8 days after transgene induction (f) and treated with the indicated compounds for the days (D) indicated (n=2 independent experiments).</p></caption><graphic xlink:href="nihms660783f8"></graphic></fig><fig id="F9" orientation="portrait" position="anchor"><label>Extended Data Figure 5</label><caption><title>Day 6 continuation analysis on day 16</title><p id="P35"><bold>a</bold>, Continuation analysis schematic. Reprogramming populations were sorted for poised (CD73<sup>high</sup> or CD49<sup>high</sup>) and non-poised (CD73<sup>low</sup>) populations on day 6, cultured for 10 days on a 3cm plate and analyzed by CyTOF on day 16. <bold>b</bold>, Morphology of CD49d<sup>high</sup>, CD73<sup>high</sup> or CD73<sup>low</sup> cells sorted on day 6 and inspected on day 10. Poised CD49d<sup>high</sup> or CD73<sup>high</sup> cells form compact colonies within several days of sorting while non-poised CD73<sup>low</sup> cells fail to do so. <bold>c</bold>, SPADE analysis (day 16) of cells sorted at day 6 for CD73<sup>high</sup>/CD49d<sup>high</sup> CD73<sup>high</sup>, CD49<sup>high</sup> and CD73<sup>low</sup> expression (continued from <xref ref-type="fig" rid="F3">Fig. 3h</xref>). Boxes highlight a SSEA1<sup>high</sup> CD326<sup>high</sup> branch that is unique to the poised populations. Colors bars (bottom) ArcSinh-transformed counts for each marker.</p></caption><graphic xlink:href="nihms660783f9"></graphic></fig><fig id="F10" orientation="portrait" position="anchor"><label>Extended Data Figure 6</label><caption><title>Continuation analysis replicates confirm a SSEA1<sup>high</sup> CD326<sup>high</sup> branch that is unique to poised populations</title><p id="P36">Continuation analysis replicates for reprogramming-prone (CD73<sup>high</sup>/CD49d<sup>high</sup>, CD73<sup>high</sup>, CD49d<sup>high</sup>) and non-prone (CD73<sup>low</sup>) populations. Boxes highlight a SSEA1<sup>high</sup> CD326<sup>high</sup> branch that is unique to the poised populations. Colors bars (bottom) represent absolute percentages (left panel) and ArcSinh-transformed counts for each marker.</p></caption><graphic xlink:href="nihms660783f10"></graphic></fig><fig id="F11" orientation="portrait" position="anchor"><label>Extended Data Figure 7</label><caption><title>Molecular characterization of reprogramming-prone intermediates</title><p id="P37"><bold>a</bold>, Genes differentially expressed between reprogramming-prone (day 6 or 9 CD73<sup>high</sup> or CD49d<sup>high</sup>) and non-prone (CD73<sup>low</sup>) populations. Genes with more than 2-fold differential expression between reprogramming-prone and non-prone were selected and k-means clustered (k=5) with control and total reprogramming population expression values. <bold>b</bold>, Heat map of pluripotency associated genes shown in <xref ref-type="fig" rid="F4">Fig. 4a</xref> (log2). <bold>c–d</bold>, Quantitative PCR verification of <italic>Etv5</italic> (c) and <italic>Nr0b1</italic> (d) expression levels (n=3 technical replicates). <bold>e–f</bold>, <italic>Etv5</italic>(e) and <italic>Nr0b1</italic>(f) knockdown qPCRs (n=3 technical replicates). <bold>g</bold>, Representative FACS plots for day 9 CD73<sup>high</sup>/CD49d<sup>high</sup> quantification shown in <xref ref-type="fig" rid="F4">figure 4c</xref>. <bold>h</bold>, Demonstration of ESC self-renewal after infection with <italic>Etv5</italic> and <italic>Nr0b1</italic> hairpins All infected ESCs continue to express <italic>Oct4</italic> after passaging except ESCs infected with sh<italic>Etv5</italic>-8 (n=1).</p></caption><graphic xlink:href="nihms660783f11"></graphic></fig><fig id="F12" orientation="portrait" position="anchor"><label>Extended Data Figure 8</label><caption><title>Characterization of high efficiency reprogramming systems</title><p id="P38"><bold>a–b</bold>, Expression analysis for CD49d, CD73 and CD104 for previously reported highly efficient reprogramming systems generated by transient expression of C/EBPα<sup><xref rid="R13" ref-type="bibr">13</xref></sup> (a) or <italic>Mbd3</italic> depletion<sup><xref rid="R12" ref-type="bibr">12</xref></sup> (b). <bold>c</bold>, <italic>Oct4-GFP</italic> transgene reporter signal and <bold>d</bold>, SSEA1 and CD326 levels for the <italic>Mbd3<sup>fl/−</sup></italic> secondary reprogramming system for untreated (left) and 9 days post induction (right) MEFs. <bold>e</bold>, SPADE analysis for reprogramming <italic>Mbd3<sup>fl/−</sup></italic> secondary MEFs at days 0, 3, 6, 9 and 12 using all surface markers. Percent total of cells and representative markers are shown for each time point. Remaining markers are shown in <xref ref-type="fig" rid="F13">Extended Data Figures 9</xref> and <xref ref-type="fig" rid="F14">10</xref>. Colors bars represent absolute percentages (left panel) and ArcSinh-transformed counts for each marker. <bold>f</bold>, Verification of <italic>Mbd3</italic> loss in passage 3 <italic>Rosa26-CreER, Mbd3<sup>fl/−</sup></italic> secondary MEFs after treatment with 4OH-tamoxifen. <italic>Mbd3</italic> levels were compared to passage 3 <italic>Rosa-rtTA<sup>+/−</sup></italic> MEFs. While there are several unspecific bands, there is clearly one band around the expected size of <italic>Mbd3</italic> absent in 4OH-tamoxifen-treated cells (arrow).</p></caption><graphic xlink:href="nihms660783f12"></graphic></fig><fig id="F13" orientation="portrait" position="anchor"><label>Extended Data Figure 9</label><caption><title>SPADE and biaxial analysis for secondary <italic>Mbd3<sup>fl/−</sup></italic> MEFs</title><p id="P39"><bold>a</bold>, SPADE analysis for 2° <italic>Mbd3<sup>fl/−</sup></italic> MEF reprogramming populations (continued from <xref ref-type="fig" rid="F12">Extended Data Fig. 8e</xref>). Colors bars (bottom) represent ArcSinh-transformed counts for each marker. <bold>b</bold>, Biaxial plots for selected markers.</p></caption><graphic xlink:href="nihms660783f13"></graphic></fig><fig id="F14" orientation="portrait" position="anchor"><label>Extended Data Figure 10</label><caption><title>SPADE analysis for secondary <italic>Mbd3<sup>fl/−</sup></italic> MEFs</title><p id="P40">SPADE analysis for 2° <italic>Mbd3<sup>fl/−</sup></italic>reprogramming populations (continued from <xref ref-type="fig" rid="F12">Extended Data Fig. 8e</xref>). Colors bars (bottom) represent ArcSinh-transformed counts for each marker.</p></caption><graphic xlink:href="nihms660783f14"></graphic></fig></sec><sec sec-type="supplementary-material" id="SM"><title>Supplementary Material</title><supplementary-material content-type="local-data" id="SD1"><label>1</label><media xlink:href="NIHMS660783-supplement-1.docx" orientation="portrait" xlink:type="simple" id="d37e1559" position="anchor"></media></supplementary-material></sec></body><back><ack id="S12"><title>Acknowledgments</title><p id="P41">We thank P. Lovelace, R. Finck, K.M. Loh, K. Tanabe and S. Marro for intellectual and technical advice. We would like to thank J. Hanna for his gift of 2° Mbd3<sup>fl/−</sup> MEFs. We would also like to thank Sebastian Knöbel at Miltenyi for his gift of mEF-SK4 antibody. This work was supported by the California Institute of Regenerative Medicine grant RB2-01592 (G.P.N.). E.L. was supported by the California Institute for Regenerative Medicine Predoctoral Fellowship TG2-01159 and National Science Foundation Graduate Research Fellowship DGE-114747. E.R.Z. was supported by NIH NRSA F32 GM093508-01. M.W. is a New York Stem Cell Foundation-Robertson Investigator and a Tashia and John Morgridge Faculty Scholar at the Child Health Research Institute at Stanford.</p></ack><fn-group><fn id="FN2" fn-type="con"><p id="P42"><bold>Author Contributions</bold></p><p id="P43">E.L., E.R.Z, G.P.N. and M.W. designed research. E.L. conducted reprogramming and sorting experiments. E.R.Z. conducted CyTOF analysis and data processing. Y.H.N. and I.N.G. assisted with sample processing. E.L., E.R.Z., G.P.N. and M.W. analyzed data. 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Color bars represent absolute percentages (top row) and ArcSinh-transformed counts for each marker. <bold>c</bold>, SPADE analysis of infected reprogramming MEF populations at days 0, 3, 6 and 9. For each marker, the same color scale was applied to every sample, allowing direct comparison between time points. Color bars represent absolute percentages (left) and ArcSinh-transformed counts.</p></caption><graphic xlink:href="nihms660783f1"></graphic></fig><fig id="F2" orientation="portrait" position="float"><label>Figure 2</label><caption><title>A surface marker screen identifies an early CD73<sup>high</sup> CD49d<sup>high</sup> reprogramming intermediate</title><p id="P46"><bold>a–c</bold>, 96 well reprogramming assays on days 3, 6 and 9. 20 cells/well sorted at days 3, 6 and 9. <italic>Sox2-EGFP</italic><sup>+</sup> colonies were assayed on day 24. Asterisks indicate two-sided t-test P-value<0.05. <bold>d</bold>, Plating efficiencies for GFP expressing MEFs sorted on day 6 for CD73<sup>high</sup> or CD49d<sup>high</sup> (assayed 24 hours post-sort). <bold>e</bold>, Single cell reprogramming efficiencies for day 6 CD73<sup>high</sup> or CD49d<sup>high</sup> fractions. <bold>f</bold>, Reprogramming efficiencies (e) adjusted for plating efficiencies (d). Error bars represent standard deviation. n=3 independent experiments for all assays.</p></caption><graphic xlink:href="nihms660783f2"></graphic></fig><fig id="F3" orientation="portrait" position="float"><label>Figure 3</label><caption><title>Characterization of CD73<sup>high</sup> and CD49d<sup>high</sup> intermediates</title><p id="P47"><bold>a–c</bold>, Percent of wells with <italic>Nanog</italic> expressing cells on day 24 for reprogramming tail tip fibroblasts (TTF) (a) (n=3) and glia (b,c) (n=1, independent primary cells and infections). <bold>d</bold>, Representative <italic>Nanog</italic> immunostaining. Scale bar is 200µm. <bold>e</bold>, Heterogeneously expressed markers in day 6 CD73<sup>high</sup> population. <bold>f</bold>, Single cell 96-well assays for day 6 CD73<sup>high</sup> fraction with additional surface markers (n=3). <bold>g</bold>, Refined poised signature. Clusters are low for mesenchymal markers. <bold>h</bold>, Continuation analysis shows SSEA1<sup>high</sup> CD326<sup>high</sup> branch unique to poised populations (boxed). All experiments represent independent biological replicates. Error bars represent standard deviation.</p></caption><graphic xlink:href="nihms660783f3"></graphic></fig><fig id="F4" orientation="portrait" position="float"><label>Figure 4</label><caption><title>Reprogramming regulators identified with CD73<sup>high</sup>/CD49d<sup>high</sup> intermediates</title><p id="P48"><bold>a</bold>, Day 6 and 9 reprogramming-prone and non-prone pluripotency associated gene differential expression. Dotted line represents value of 1(no difference). <bold>b–c</bold>, Day 9 CD73<sup>high</sup>/CD49d<sup>high</sup> quantification for knockdown (b, n=3 independent experiments) and rescue (c) experiments. Gating shown in <xref ref-type="fig" rid="F11">Extended Data Fig. 7g</xref>. Asterisks indicate two-sided t-test P-value<0.05. <bold>d–e</bold>, Day 24 <italic>Sox2-EGFP</italic><sup>+</sup>(d, n=1) and <italic>Nanog</italic><sup>+</sup> colonies (e, n=2 independent experiments) from <italic>Rosa-rtTA<sup>+/−</sup>, Sox2-EGFP<sup>+/−</sup></italic> and <italic>Rosa-rtTA<sup>+/−</sup></italic> MEFs, respectively. <bold>f</bold>, Early and late reprogramming model. Dotted red boxes distinguish CD104 observed in the <italic>Mbd3<sup>fl/−</sup></italic> system. Error bars indicate standard deviation.</p></caption><graphic xlink:href="nihms660783f4"></graphic></fig></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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