2024-03-28T19:30:01Zhttps://www.tdx.cat/oai/requestoai:www.tdx.cat:10803/2834842024-03-15T10:57:29Zcom_10803_236col_10803_690278
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Stem cells
Cell reprogramming
iPS cells
Pluripotent stem cells
Embryonic stem cells
Transdifferentiation
Hematopoiesi
Células madre
Reprogramación celular
Células iPS
Células madre pluripotentes
Células madre embrionaria
Transdiferenciación
Hematopoyeisis
C/EBPα poises B cells for rapid reprogramming into induced pluripotent stem cells
[Barcelona] :
Universitat Pompeu Fabra,
2014
Accés lliure
http://hdl.handle.net/10803/283484
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Di Stefano, Bruno,
autor
Programa de doctorat en Biomedicina,
degree
1 recurs en línia (116 pàgines)
Tesi
Doctorat
Universitat Pompeu Fabra. Departament de Ciències Experimentals i de la Salut
2014
Universitat Pompeu Fabra. Departament de Ciències Experimentals i de la Salut
Tesis i dissertacions electròniques
Graf, T.
(Thomas)
supervisor acadèmic
TDX
One of the major goals of current stem cell research is understanding the
mechanism of somatic cell reprogramming by Oct4, Sox2, Klf4 and Myc
(OSKM) into induced pluripotent stem cells (iPSCs). However, the finding that
only a small proportion of the cells become reprogrammed, typically requiring
>12 days, has hampered progress towards this goal.
C/EBPα is a transcription factor specifically expressed in myelomonocytic
cells within the hematopoietic system whose forced expression in B cells
efficiently induces transdifferentiation into macrophages. We have now found
that an 18-hour pulse of C/EBPα expression followed by OSKM activation
induces an approximately 100-fold increase in the iPSC reprogramming
efficiency, involving up to 95% of the cells within a week. Concomitantly, the
cells undergo an epithelial-mesenchymal transition and pluripotency genes
become upregulated to levels comparable to embryonic stem and iPS cells.
In serum-free conditions the process is further accelerated, with 60% of the
poised and OSKM induced B cells becoming Oct4-GFP positive within 2 days.
These results are consistent with the idea that the C/EBPα pulse helps to
overcome the stochastic phase of iPSC reprogramming. In addition, our work
shed new light on the role of C/EBPα in induced pluripotency. Our data
indicate that C/EBPα acts as a pathbreaker, at least in part mediated by the
dioxygenase Tet2. C/EBPα binds to the Tet2 gene, induces its expression
and translocates the protein to the nucleus. Here Tet2 binds to regulatory
regions of pluripotency genes and converts methylated cytosine residues into
hydroxymethylated cytosines. The pulse also renders the chromatin at
regulatory sites of pluripotency genes accessible to DNase I digestion and,
following OSKM induction, leads to local demethylation and to the binding of
Oct4, correlating with the observed rapid upregulation of pluripotency genes.
In line with an important role of Tet2 as a mediator of reprogramming, coexpression
of the gene with OSKM enhanced B cell reprogramming
substantially. The rapid and highly efficient iPSC reprogramming approach
described herein should help to fully elucidate the early events of
reprogramming to pluripotency and, if applicable to human cells, could have
potential clinical applications.
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