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Early reprogramming regulators identified by prospective isolation and mass cytometry

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(1–11). Despite recent reports claiming substantially increased reprogramming efficiencies u...

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Autores principales: Lujan, Ernesto, Zunder, Eli R., Ng, Yi Han, Goronzy, Isabel N., Nolan, Garry P., Wernig, Marius
Formato: Online Artículo Texto
Lenguaje:English
Publicado: 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4441548/
https://www.ncbi.nlm.nih.gov/pubmed/25830878
http://dx.doi.org/10.1038/nature14274
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author Lujan, Ernesto
Zunder, Eli R.
Ng, Yi Han
Goronzy, Isabel N.
Nolan, Garry P.
Wernig, Marius
author_facet Lujan, Ernesto
Zunder, Eli R.
Ng, Yi Han
Goronzy, Isabel N.
Nolan, Garry P.
Wernig, Marius
author_sort Lujan, Ernesto
collection PubMed
description 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(1–11). Despite recent reports claiming substantially increased reprogramming efficiencies using genetically modified donor cells(12,13) 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(1,2,7,8,10). 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(2). Expression profiling revealed early upregulation of the transcriptional regulators Nr0b1 and Etv5 in this reprogramming state, preceding activation of key pluripotency regulators such as Rex1, Dppa2, Nanog and Sox2. 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.
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spelling pubmed-44415482015-11-21 Early reprogramming regulators identified by prospective isolation and mass cytometry Lujan, Ernesto Zunder, Eli R. Ng, Yi Han Goronzy, Isabel N. Nolan, Garry P. Wernig, Marius Nature Article 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(1–11). Despite recent reports claiming substantially increased reprogramming efficiencies using genetically modified donor cells(12,13) 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(1,2,7,8,10). 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(2). Expression profiling revealed early upregulation of the transcriptional regulators Nr0b1 and Etv5 in this reprogramming state, preceding activation of key pluripotency regulators such as Rex1, Dppa2, Nanog and Sox2. 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. 2015-04-01 2015-05-21 /pmc/articles/PMC4441548/ /pubmed/25830878 http://dx.doi.org/10.1038/nature14274 Text en http://www.nature.com/authors/editorial_policies/license.html#terms 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:http://www.nature.com/authors/editorial_policies/license.html#terms
spellingShingle Article
Lujan, Ernesto
Zunder, Eli R.
Ng, Yi Han
Goronzy, Isabel N.
Nolan, Garry P.
Wernig, Marius
Early reprogramming regulators identified by prospective isolation and mass cytometry
title Early reprogramming regulators identified by prospective isolation and mass cytometry
title_full Early reprogramming regulators identified by prospective isolation and mass cytometry
title_fullStr Early reprogramming regulators identified by prospective isolation and mass cytometry
title_full_unstemmed Early reprogramming regulators identified by prospective isolation and mass cytometry
title_short Early reprogramming regulators identified by prospective isolation and mass cytometry
title_sort early reprogramming regulators identified by prospective isolation and mass cytometry
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4441548/
https://www.ncbi.nlm.nih.gov/pubmed/25830878
http://dx.doi.org/10.1038/nature14274
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