329
Using Xenopus to Understand Pluripotency
formative step in the path to manipulation of pluripotency and
cell fate reprogramming, with the generation of induced pluripotent stem cells (iPSCs) ( Takahashi and Yamanaka, 2006 ).
In a strictly in vitro setting, the over-expression of a certain
set of pioneer transcription factors can reprogram differentiated cells to an induced pluripotent state ( Figure 23.1 ). iPS
cells generated in this way are in many aspects functionally
equivalent to ES cells. Due to its ease of implementation, the
iPS procedure has advanced the journey towards cell replacement to a great extent. Nevertheless, the generation of iPSCs
via transcription factor over-expression remains ineff cient
and stochastic. In addition, it is argued that the quality of
ES-cells derived from NT-embryos is higher than of iPSCs, as
measured by chromosomal abnormalities and differentiation
potential ( Matoba and Zhang, 2018 ).
The early advances in nuclear reprogramming procedures
were further complemented by the discovery of transdifferentiation—a technique that aims at reprogramming the cell
from its inherent differentiated type directly to another differentiated cell type, all while surpassing the dedifferentiation process that is key in NT. For example, in one of the
frst transdifferentiation experiments, ectopic expression of
the pioneer transcription factor MyoD by transfection of its
cDNA into mouse fbroblasts converted the cells into myoblasts ( Davis et al., 1987 ).
While the mechanisms of cell fate reprogramming may
vary among these different techniques, what they all have in
common is that differentiated cells subjected to these reprogramming methods show resistance, and reprogramming
effciencies are low. In the past, using NT and Xenopus laevis
as model system, key mechanisms of nuclear reprogramming
were revealed, and initial insights into the mechanistic basis
of resistance to reprogramming were gained. Importantly,
the obtained results were confrmed in mammalian reprogramming systems and hence signifcantly advanced the
entire feld of nuclear reprogramming, as summarized next.
23.4. MOLECULAR INSIGHTS INTO THE
PROCESS OF NUCLEAR REPROGRAMMING
GAINED IN XENOPUS LAEVIS
The last few decades of the 1900s witnessed a lag in the use
of SCNT to study the resistance of differentiated cell types
against reprogramming. Spanning amphibians to mammals,
the effciency of cloning was consistently low, and absence
of the high-throughput technology available today limited
further work on this problem. Developments in molecular
cloning, refned tissue culture methods, and other molecular
biology techniques were used hand in hand with the alternative reprogramming technologies discussed previously.
However, while these methods broadened the scope of the
feld, the issue of resistance against reprogramming in differentiated cells was universal, with little headway into the reasons causing this. Whether in vivo enucleated oocytes or in
vitro egg extracts, there existed a pervading struggle between
the reprogramming activities of the egg and the mechanisms
maintaining stable cell fates of the differentiated nucleus—a
“battle for supremacy,” as termed by John Gurdon. In the
21st century, the reprogramming feld was revived with new
vigor with the onset of genomic and proteomic techniques.
The development of transcriptional prof ling techniques,
histone modif cation-specifc proteomic tools, and, later,
sequencing technology was key to driving the current era
of the reprogramming feld and led to important discoveries
concerning resistance to reprogramming.
23.4.1. REPROGRAMMING AND DNA REPLICATION
One evident incongruity in the development of a NT embryo
is the frequency of cell division of an adult cell versus that
of the donor nucleus in SCNT experiments. Due to the rather
quiescent cell division of an adult cell, the extremely rapid
cell divisions enforced on the donor nucleus during NT may
cause severe replication defects, limiting the probability of
such a cell to give rise to a fully developed, fertile organism. During SCNT, microinjection causes activation of the
oocyte. As the egg divides 90 minutes after activation, the
DNA synthesis within the transplanted nucleus—a process
which normally requires approximately six hours in somatic
cells—must be acquired within this time frame upon NT
to the egg. This transition from slow to rapid DNA replication can take a toll on the nucleus, causing DNA damage
or a lag in DNA replication. Thus, occasionally, the entire
replicating genome may move into only one of the dividing
blastomeres, leading to a partially cleaved blastula. Previous
attempts to increase reprogramming effciency of adult cells
using “serial nuclear transfers”—NT is repeated using a partial or complete blastula cell nucleus derived from a developing NT embryo—have proved substantially successful (King
and Briggs, 1956; Gurdon et al., 1958; Laskey and Gurdon,
1970). It is thought that a subsequent NT event provides the
embryo a second opportunity to complete DNA replication,
which allows it to develop much further. Studies in Xenopus
egg extracts confrmed that differentiated erythrocyte nuclei
indeed replicate ineffciently in interphase Xenopus egg
extracts when compared to sperm nuclei, and this difference
in replication effciency decreases when differentiated nuclei
are allowed to progress through mitosis frst (Lemaitre et al.,
2005). Each additional round of mitosis furthers the capability of differentiated nuclei to undergo DNA replication
upon NT. Thus, remodeling of the chromatin, which occurs
at each embryonic metaphase, may support reprogramming
of replicon organization in NT embryos to an early embryonic state and so permit successful development in some
instances. However, some somatic nuclei, such as spermatid nuclei, show effcient reprogramming of replication origins comparable to those of sperm nuclei (Teperek et al.,
2016). While sperm nuclei almost always support successful
embryonic development, spermatids do not and show just as
low developmental potential as other somatic nuclei when
transplanted to eggs to generate NT embryos. In summary,
this suggests that an adjustment of the replication machinery
to an embryonic state is likely benefcial for nuclear reprogramming and also that additional layers of resistance exist
Using Xenopus to Understand Pluripotency
formative step in the path to manipulation of pluripotency and
cell fate reprogramming, with the generation of induced pluripotent stem cells (iPSCs) ( Takahashi and Yamanaka, 2006 ).
In a strictly in vitro setting, the over-expression of a certain
set of pioneer transcription factors can reprogram differentiated cells to an induced pluripotent state ( Figure 23.1 ). iPS
cells generated in this way are in many aspects functionally
equivalent to ES cells. Due to its ease of implementation, the
iPS procedure has advanced the journey towards cell replacement to a great extent. Nevertheless, the generation of iPSCs
via transcription factor over-expression remains ineff cient
and stochastic. In addition, it is argued that the quality of
ES-cells derived from NT-embryos is higher than of iPSCs, as
measured by chromosomal abnormalities and differentiation
potential ( Matoba and Zhang, 2018 ).
The early advances in nuclear reprogramming procedures
were further complemented by the discovery of transdifferentiation—a technique that aims at reprogramming the cell
from its inherent differentiated type directly to another differentiated cell type, all while surpassing the dedifferentiation process that is key in NT. For example, in one of the
frst transdifferentiation experiments, ectopic expression of
the pioneer transcription factor MyoD by transfection of its
cDNA into mouse fbroblasts converted the cells into myoblasts ( Davis et al., 1987 ).
While the mechanisms of cell fate reprogramming may
vary among these different techniques, what they all have in
common is that differentiated cells subjected to these reprogramming methods show resistance, and reprogramming
effciencies are low. In the past, using NT and Xenopus laevis
as model system, key mechanisms of nuclear reprogramming
were revealed, and initial insights into the mechanistic basis
of resistance to reprogramming were gained. Importantly,
the obtained results were confrmed in mammalian reprogramming systems and hence signifcantly advanced the
entire feld of nuclear reprogramming, as summarized next.
23.4. MOLECULAR INSIGHTS INTO THE
PROCESS OF NUCLEAR REPROGRAMMING
GAINED IN XENOPUS LAEVIS
The last few decades of the 1900s witnessed a lag in the use
of SCNT to study the resistance of differentiated cell types
against reprogramming. Spanning amphibians to mammals,
the effciency of cloning was consistently low, and absence
of the high-throughput technology available today limited
further work on this problem. Developments in molecular
cloning, refned tissue culture methods, and other molecular
biology techniques were used hand in hand with the alternative reprogramming technologies discussed previously.
However, while these methods broadened the scope of the
feld, the issue of resistance against reprogramming in differentiated cells was universal, with little headway into the reasons causing this. Whether in vivo enucleated oocytes or in
vitro egg extracts, there existed a pervading struggle between
the reprogramming activities of the egg and the mechanisms
maintaining stable cell fates of the differentiated nucleus—a
“battle for supremacy,” as termed by John Gurdon. In the
21st century, the reprogramming feld was revived with new
vigor with the onset of genomic and proteomic techniques.
The development of transcriptional prof ling techniques,
histone modif cation-specifc proteomic tools, and, later,
sequencing technology was key to driving the current era
of the reprogramming feld and led to important discoveries
concerning resistance to reprogramming.
23.4.1. REPROGRAMMING AND DNA REPLICATION
One evident incongruity in the development of a NT embryo
is the frequency of cell division of an adult cell versus that
of the donor nucleus in SCNT experiments. Due to the rather
quiescent cell division of an adult cell, the extremely rapid
cell divisions enforced on the donor nucleus during NT may
cause severe replication defects, limiting the probability of
such a cell to give rise to a fully developed, fertile organism. During SCNT, microinjection causes activation of the
oocyte. As the egg divides 90 minutes after activation, the
DNA synthesis within the transplanted nucleus—a process
which normally requires approximately six hours in somatic
cells—must be acquired within this time frame upon NT
to the egg. This transition from slow to rapid DNA replication can take a toll on the nucleus, causing DNA damage
or a lag in DNA replication. Thus, occasionally, the entire
replicating genome may move into only one of the dividing
blastomeres, leading to a partially cleaved blastula. Previous
attempts to increase reprogramming effciency of adult cells
using “serial nuclear transfers”—NT is repeated using a partial or complete blastula cell nucleus derived from a developing NT embryo—have proved substantially successful (King
and Briggs, 1956; Gurdon et al., 1958; Laskey and Gurdon,
1970). It is thought that a subsequent NT event provides the
embryo a second opportunity to complete DNA replication,
which allows it to develop much further. Studies in Xenopus
egg extracts confrmed that differentiated erythrocyte nuclei
indeed replicate ineffciently in interphase Xenopus egg
extracts when compared to sperm nuclei, and this difference
in replication effciency decreases when differentiated nuclei
are allowed to progress through mitosis frst (Lemaitre et al.,
2005). Each additional round of mitosis furthers the capability of differentiated nuclei to undergo DNA replication
upon NT. Thus, remodeling of the chromatin, which occurs
at each embryonic metaphase, may support reprogramming
of replicon organization in NT embryos to an early embryonic state and so permit successful development in some
instances. However, some somatic nuclei, such as spermatid nuclei, show effcient reprogramming of replication origins comparable to those of sperm nuclei (Teperek et al.,
2016). While sperm nuclei almost always support successful
embryonic development, spermatids do not and show just as
low developmental potential as other somatic nuclei when
transplanted to eggs to generate NT embryos. In summary,
this suggests that an adjustment of the replication machinery
to an embryonic state is likely benefcial for nuclear reprogramming and also that additional layers of resistance exist