Early reprogramming regulators identified by prospective isolation and mass cytometry
Identifieur interne : 000398 ( Pmc/Curation ); précédent : 000397; suivant : 000399Early reprogramming regulators identified by prospective isolation and mass cytometry
Auteurs : Ernesto Lujan [États-Unis] ; Eli R. Zunder [États-Unis] ; Yi Han Ng [États-Unis] ; Isabel N. Goronzy [États-Unis] ; Garry P. Nolan [États-Unis] ; Marius Wernig [États-Unis]Source :
- Nature [ 0028-0836 ] ; 2015.
Abstract
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
Url:
DOI: 10.1038/nature14274
PubMed: 25830878
PubMed Central: 4441548
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<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>
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<article-categories><subj-group subj-group-type="heading"><subject>Article</subject>
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<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>
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</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>
<floats-group><fig id="F1" orientation="portrait" position="float"><label>Figure 1</label>
<caption><title>Reprogramming surface marker profiling by mass cytometry</title>
<p id="P45"><bold>a</bold>
, Histogram overlays show enrichment levels for the three cell populations analyzed: mouse embryonic fibroblasts (MEF, black), partially reprogrammed cells (PRC, red) and embryonic stem cells (ESC, green). <bold>b</bold>
, SPADE analysis of combined MEF, PRC and ESC datasets. 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>
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