4122 Research Article
Introduction
It has been suggested that the fusion of embryonic stem cells (ESCs)
with somatic cells is a unidirectional process giving rise to hybrid
cells that retain the phenotype of ESCs but not that of somatic cells
(Silva et al., 2006). Therefore, the somatic cell state seems to be a
nonviable state after cell fusion. The phenotypical characteristics
of ESC–somatic-cell fusion-hybrid cells include the morphological
features of the pluripotent fusion partner as well as a specific
epigenetic state and gene-expression profile; inactivation of tissue-
specific genes expressed in the somatic cell-fusion partner; and a
differentiation potential characteristic of ESCs (Do and Scholer,
2006). Investigations into the characteristics of the hybrids formed
by the fusion of embryonic germ cells (EGCs) or embryonal
carcinoma cells (ECCs) with somatic cells have shown that EGCs
and ECCs also have the capacity to reprogram somatic cells to the
pluripotent phenotype (Do et al., 2007; Tada et al., 1997). Thus,
studies investigating the fusion of pluripotent cells with somatic
cells have consistently demonstrated that the reprogramming process
is unidirectional. That is, pluripotent cells effectively reprogram
somatic cells, abolishing the gene-expression patterns of the somatic
cell and resetting the gene-expression profile to that of the
pluripotent state. However, the gene-expression profile of the
fusion-hybrid cells is similar, but not identical, to that of
the pluripotent cell-fusion partner, indicating that fusion-induced
reprogramming is a unidirectional process that occurs concomitantly
with the incomplete reprogramming of certain genes (Ambrosi et
al., 2007). Therefore, fusion-induced reprogramming does not
necessarily result in the complete genetic reprogramming of somatic
cells to a phenotype identical to that of the pluripotent cell-fusion
partner. Rather, the fusion-hybrid cells might still retain residual
‘memory’ of the somatic cells, although this somatic memory might
not affect the characteristics of the pluripotent cell-fusion partner
of the pluripotent fusion-hybrid cells. Moreover, it is still possible
that fusion-induced reprogramming is not a solely unidirectional
process, because somatic cells might be capable of inducing
alterations in the characteristics of their pluripotent cell-fusion
partners. Here, we challenged the dogma that fusion-induced
pluripotential reprogramming is a unidirectional process and we
assessed the possibility that somatic cells can also induce alterations
in the characteristics of their pluripotent cell-fusion partners.
Results
Fusion between ECCs and ESCs results in two different types
of hybrid cells
As a first step in investigating whether fusion-induced
reprogramming is solely unidirectional, we fused ESCs with ECCs
– two different types of pluripotent cells with the potential to
reprogram somatic cells (Do et al., 2007; Do and Scholer, 2004).
Specifically, we fused OG2 ESCs (containing an Oct4-GFP
transgene) with -geoF9 ECCs (hereafter referred to as F9 ECCs;
containing a neo/lacZ transgene) to obtain selectable fusion-hybrid
cells. On day 2 post-fusion, GFP-positive cells were sorted by
fluorescence-activated cell sorting (FACS) and cultured in ESC
medium containing G418 on feeder layers. After 10 days in culture
with selection medium, the majority of cells had formed ECC-like
colonies (141/143; 98.6%), but a small minority had formed ESC-
like colonies (2/143; 1.4%) (Fig. 1A). ECC- and ESC-like hybrid
cells were cloned and cultured on gelatin-coated dishes or on feeder
The fusion of somatic cells with pluripotent cells results in the
generation of pluripotent hybrid cells. Because the ‘memory’
of somatic cells seems to be erased during fusion-induced
reprogramming, genetic reprogramming is thought to be a
largely unidirectional process. Here we show that fusion-
induced reprogramming, which brings about the formation of
pluripotent hybrids, does not always follow a unidirectional
route. Xist is a unique gene in that it is reprogrammed to the
state of somatic cells in fusion-induced pluripotent hybrids. In
hybrids formed from the cell fusion of embryonal carcinoma
cells (ECCs) with male neural stem cells (mNSCs), the Xist gene
was found to be reprogrammed to the somatic cell state,
whereas the pluripotency-related and tissue-specific marker
genes were reprogrammed to the pluripotent cell state.
Specifically, Xist is not expressed in hybrids, because the
‘memory’ of the somatic cell has been retained (i.e. mNSCs do
not exhibit Xist expression) and that of the pluripotent cell
erased (i.e. inactivation of the partially active Xist gene of ECCs,
complete methylation of the Xist region). The latter phenomenon
is induced by male, but not by female, NSCs.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/122/22/4122/DC1
Key words: Reprogramming, Cell fusion, Pluripotency, Oct4, Xist
Summary
Reprogramming of Xist against the pluripotent state in
fusion hybrids
Jeong Tae Do1,*, Dong Wook Han2, Luca Gentile2, Ingeborg Sobek-Klocke2, Anton Wutz3
and Hans R. Schöler2
1Laboratory of Stem Cell and Developmental Biology, CHA Stem Cell Institute, CHA University, 605-21 Yoeksam 1-dong, Gangnam-gu, Seoul 135-
081, Korea
2Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Röntgenstrasse 20, 48149 Münster, Germany
3Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, 1030 Vienna, Austria
*Author for correspondence (dojt@cha.ac.kr)
Accepted 4 September 2009
Journal of Cell Science 122, 4122-4129 Published by The Company of Biologists 2009
doi:10.1242/jcs.056119
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4123Reprogramming of Xist against pluripotency
layers, respectively. ECC- and ESC-like hybrid cells stained positive
for both GFP (derived from an OG2 ESC) and X-gal (derived from
an F9 ECC). In addition, cells making up the ECC- and ESC-like
colonies were tetraploid in nature (Fig. 1B), indicating that they
were all ECC-ESC fusion-hybrid cells. Therefore, the vast majority
of hybrids produced by the fusion of pluripotent ECCs with ESCs
were reprogrammed morphologically to ECCs and only a minority
to ESCs. In addition, the differentiation potential of ECC- and ESC-
like hybrid cells was similar to that of F9 ECCs (rarely
differentiating into cells of neuronal lineage) and ESCs, respectively
(Fig. 2A,B). In the presence of retinoic acid (RA), F9 ECCs
preferentially differentiate into cells of primitive or parietal
endoderm but not to those of the neural lineage (Do et al., 2007).
Therefore, the poor neural-differentiation potential of the ECC-like
hybrid cells suggested that they might be a suitable model to
investigate whether ECC-like hybrid cells exhibit the differentiation
characteristics of F9 ECCs. Therefore, we assayed the ECC-like
hybrid cells for the presence of an endodermal marker (Sox17) and
an ectodermal marker (Tuj1), because F9 cells normally do not
differentiate into cells of the neuronal lineage (Tuj1-positive cells).
As expected, ECC-like hybrid cells preferentially differentiated into
endodermal cells (8165±1063/10,000 FACS-sorted cells), similar
to the ESC-like hybrid cells (2038±312/10,000 FACS-sorted cells).
Moreover, ECC-like hybrid cells rarely differentiated into neurons
(37±12/10,000 FACS-sorted cells), similar to the ESC-like hybrid
cells (3257±10,000 FACS-sorted cells).
Fig. 1. Characterization of ECC-ESC
hybrid cells. (A)Hybrid cells form
two different types of colonies (ECC-
like and ESC-like) on feeder layers.
(B)ECC-like and ESC-like hybrid
cells were observed to stain positive
for both GFP (OG2-ESC derived) and
X-gal (-geoF9-ECC derived), and to
exhibit tetraploidy. Scale bars: 25m.
(C)Real-time RT-PCR analysis of the
hybrid cells for pluripotency-related
and germ-cell-marker gene expression.
y-axis values are on a logarithmic
scale, and each minor gridline is one
tenth the value of each major gridline.
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