|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 21 May 2008
doi: 10.1242/dev.015453
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Review |
1 Center for Reproductive Medicine, Academic Medical Center, University of
Amsterdam, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands.
2 Department of Endocrinology and Metabolism, Faculty of Science, Utrecht
University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
* Author for correspondence (e-mail: d.g.derooij{at}uu.nl)
SUMMARY
In recent years, embryonic stem (ES) cell-like cells have been obtained from cultured mouse spermatogonial stem cells (SSCs). These advances have shown that SSCs can transition from being the stem cell-producing cells of spermatogenesis to being multipotent cells that can differentiate into derivatives of all three germ layers. As such, they offer new possibilities for studying the mechanisms that regulate stem cell differentiation. The extension of these findings to human SSCs offers a route to obtaining personalized ES-like or differentiated cells for use in regenerative medicine. Here, we compare the different approaches used to derive ES-like cells from SSCs and discuss their importance to clinical and developmental research.
Introduction
Spermatogonial stem cell (SSC) research has experienced an enormous boost
since 1994, when a functional assay for these cells was first developed
(Brinster and Avarbock, 1994
;
Brinster and Zimmermann, 1994).
More recently, culture systems have been developed for the long-term culture
and in vitro propagation of SSCs
(Kanatsu-Shinohara et al.,
2003; Kanatsu-Shinohara et
al., 2005; Kubota and
Brinster, 2006), which have lead to the discovery that SSCs can be
induced to become multipotent cells again, able to differentiate into various
differentiated cell lineages. In fact, these multipotent cells resemble
embryonic stem (ES) cells in their differentiation capacity and in the
morphology and growth characteristics of the colonies they form in culture,
and, as such, in this review they will be referred to as ES-like cells. These
findings have brought excitement and enthusiasm to the field, and several
reports have commented on the possible uses of these SSC-derived ES-like
cells, including in regenerative medicine
(Cyranoski, 2006;
de Rooij, 2006;
Kanatsu-Shinohara and Shinohara,
2006; Nayernia,
2007; Nayernia,
2008). However, the ways in which SSCs are transformed into
ES-like cells and how these cells are induced to differentiate differ
considerably between the various groups that are studying SSCs.
In this review, we describe and compare the methods that these groups have used and the results they have obtained, with the aim of informing developmental researchers about the usefulness of these various methods in an otherwise rather confusing and complex field. We describe how recent results show that SSCs can be propagated in culture and discuss how SSCs will enable studies into the mechanisms that govern how stem cells that are already dedicated to a specific lineage can dedifferentiate and return to a multipotent state. We also discuss how they can transdifferentiate into stem cells of another lineage.
In articles on the production of ES-like cells from SSCs, each group has used different names for the cultured SSCs and ES-like cells that have been formed. For the sake of clarity, we use only the latter terms in this review. All data available to date on the derivation of ES-like cells from SSCs derive from experiments in mice; these data will therefore be interpreted in the context of our current knowledge of spermatogonial multiplication and stem cell renewal in rodents.
Spermatogonial stem cells
SSCs reside on the basal membrane of the seminiferous tubules and are
single cells (Fig. 1). Upon
their division, the daughter cells can move away from each other to lead to
stem cell renewal, or they can stay together as a pair of so-called
Apr spermatogonia that are connected by an intercellular
cytoplasmic bridge (Fig. 2).
This latter event represents the first step in the differentiation process
that ultimately leads to the formation of spermatozoa. After the formation of
a pair of Apr spermatogonia, there are nine to ten further
divisions, which lead to spermatogonial clones of increasing length (see Figs
1 and
2). Then, spermatocytes form
that leave the basal membrane and take up a position closer to the lumen of
the tubules. Although it was recently suggested that pairs and short chains of
spermatogonia can also have stem cell properties
(Nakagawa et al., 2007), this
is still the prevailing scheme of spermatogonial multiplication and stem cell
renewal (for a review, see de Rooij,
2001).
SSCs can be morphologically distinguished in whole-mounts of seminiferous
tubules and can be counted (e.g.
Tegelenbosch and de Rooij,
1993), but since 1994 (Brinster
and Avarbock, 1994; Brinster
and Zimmermann, 1994) it has also become possible to determine the
presence of stem cells by a functional test, the SSC transplantation assay. In
this assay, cell suspensions that contain SSCs are transplanted into the
testes of recipient mice, in which endogenous spermatogenesis has been
abolished, for example, by treating them with a cytostatic drug or by
irradiation (Brinster and Avarbock,
1994; Brinster and Zimmermann,
1994; Creemers et al.,
2002). This method makes it possible to detect the presence of
functional stem cells in a cell suspension and to compare stem cell numbers
after various treatments or culture periods. Recently, a much less laborious
assay, based on the in vitro formation of SSC colonies, has been developed
(Yeh et al., 2007).
The isolation of SSCs
The first step when working with SSCs in vitro is to isolate these cells.
In the mouse, only about 0.03% of the germ cells are stem cells
(Tegelenbosch and de Rooij,
1993). Therefore, an attempt is usually made to purify SSCs from
isolated germ cells. Surprisingly, the various groups working on the
plasticity of SSCs have used widely different SSC purification approaches.
Kanatsu-Shinohara et al.
(Kanatsu-Shinohara et al.,
2004), for example, have used the testes of neonatal mice to
purify SSCs, in which the only germ cell types present are the earliest types
of spermatogonia, including SSCs. Guan et al.
(Guan et al., 2006) have used
testes of 4- to 6-week-old mice as the starting material, which have the full
range of germ cell types, and have purified SSCs from this tissue by sorting
for cells expressing STRA8 (stimulated by retinoic acid gene 8). As STRA8 is
expressed by all premeiotic cells, this selection is for all types of
spermatogonia, which will render a less pure population of SSCs than that
obtained by Kanatsu-Shinohara et al.
(Kanatsu-Shinohara et al.,
2004). Seandel et al. (Seandel
et al., 2007) isolated a cell suspension that contains SSCs by
isolating germ cells from testes of 3- to 35-week-old mice. Clearly, this
procedure renders a much less pure SSC population than that which uses
neonatal mouse testes. Hu et al. (Hu et
al., 2007) isolated germ cells from 6- to 8-day-old mouse testes.
As these testes only contain spermatogonia and possibly early spermatocytes,
the degree of purity of the cells obtained is comparable to that obtained by
Guan et al. Finally, Boulanger et al.
(Boulanger et al., 2007)
removed the interstitial cells from seminiferous tubules prepared from adult
mouse testes and just made a cell suspension of the tubules.
|
|
1,
one of the receptors for glial cell line derived neurotrophic factor (GDNF).
GDNF is produced by Sertoli cells and is probably the most important growth
factor involved in the regulation of SSC renewal and proliferation
(Meng et al., 2000).
GFR1 is only expressed in SSCs and in the pairs of spermatogonia that
result from the differentiating divisions of these cells, and therefore the
percentage of SSCs must be high in the cell suspensions sorted for its
expression. Hofmann et al. and Buageaw et al. showed the feasibility of SSC
purification by making use of GFR1 as a membrane marker, but no figures
for the efficiency of the purification were given
(Hofmann et al., 2005;
Buageaw et al., 2005). In all
events, the conclusion of all these results must be that the purity of the
starting population of SSCs is not of crucial importance for producing ES-like
cells. The culture and induction of ES-like colonies from SSCs
Kanatsu-Shinohara et al. cultured SSCs in such a way that these cells
propagated themselves, while retaining their capacity to repopulate a
recipient mouse testis upon transplantation
(Kanatsu-Shinohara et al.,
2004). A special medium was used, designed to culture
hematopoietic stem cells, to which several growth factors, including GDNF,
were added. In this culture system, a feeder layer is first formed that is
composed of the contaminating somatic cells of the neonatal testis. Then,
after 2 weeks and two passages, mitomycin-treated mouse embryonic fibroblasts
(MEFs) are used as a feeder layer. During the first weeks of culture, the only
colonies that formed consisted of SSCs, but, within 4-7 weeks, colonies formed
that morphologically resembled ES cell colonies. Further work indicated that
these colonies were indeed composed of multipotent ES-like cells. In order to
maintain the multipotent character of these ES-like cells, they subsequently
had to be cultured under standard ES cell culture conditions in medium
containing 15% fetal calf serum and LIF. Under these conditions, the cultured
SSCs could not be propagated because of the lack of GDNF. ES-like colonies
could only be obtained when the starting population of SSCs was derived from
neonatal mice; when it was derived from older mice, ES-like colonies did not
appear. However, cultures of SSCs derived from adult p53
(Trp53)-null mice did produce ES-like colonies. P53 is involved in
the cellular response to DNA damage and a lack of P53 increases the chances of
teratoma development. Possibly, P53-deficient SSCs are more capable of
undergoing the transition into ES-like cells.
An essentially similar protocol was followed by Seandel et al.
(Seandel et al., 2007), except
that this group used inactivated testicular stromal cells consisting of a
mixture of CD34+ peritubular cells,
-smooth-muscle-actin-positive peritubular cells and cells positive for
the Sertoli cell marker vimentin, as a feeder layer because they had less
success using MEFs. By this method, ES-like colonies only appeared after more
than 3 months in culture, more slowly than reported by Kanatsu-Shinohara et
al. (Kanatsu-Shinohara et al.,
2004). A substantially different approach was taken by Guan et al.
(Guan et al., 2006). Their
starting material was derived from 4- to 6-week-old mice and they did not use
the stem cell medium described by Kanatsu-Shinohara et al.
(Kanatsu-Shinohara et al.,
2004) but simply Dulbecco's Modified Eagle's Medium (DMEM) with
serum and added GDNF, in which testicular cells were initially cultured for 4
to 7 days. These cells were then sorted for the expression of STRA8 and
subsequently cultured in DMEM under various conditions, but without adding
GDNF. Colonies of ES-like cells formed when LIF was added to the medium and/or
when the cells were cultured on a feeder layer of MEFs. The ES-like cells were
further expanded by culture on MEFs and added LIF.
Hu et al. (Hu et al., 2007)
cultured germ cells of prepubertal mice under conditions that favor osteoblast
differentiation and reported the emergence of cells that had characteristics
of osteoblasts after several weeks in culture. In this system, there was no
period of culture with added GDNF. Finally, Boulanger et al.
(Boulanger et al., 2007)
employed no culture step at all. This group transplanted cells isolated from
adult mouse seminiferous tubules, together with mammary cells, into mammary
fat pads to obtain the differentiation of SSCs into mammary epithelial
cells.
Taken together, it does not seem that a very specific approach is required
to obtain the transformation of SSCs into ES-like cells (see
Table 1). This transformation
can occur on different feeder layers and even without a feeder layer, provided
that LIF is added to the culture medium. Furthermore, the culture medium also
does not seem to play a decisive role in the transformation of SSCs into
ES-like cells, as the groups of Kanatsu-Shinohara et al.
(Kanatsu-Shinohara et al.,
2004) and Seandel et al.
(Seandel et al., 2007) used a
specific stem cell medium, whereas Guan et al.
(Guan et al., 2006) used DMEM.
All three groups did add GDNF to the culture, either continuously
(Kanatsu-Shinohara et al.,
2004; Seandel et al.,
2007) or only at the start
(Guan et al., 2006). However,
to obtain the transformation of SSCs into cells of another lineage, it might
not be necessary for them to become ES-like cells first. Putting the SSCs in
an osteoblast-inductive environment in culture
(Hu et al., 2007) or
transplanting them into a mammary gland-inductive environment in vivo
(Boulanger et al., 2007) might
be enough for these cells to change their lineage. This rather suggests that
SSCs are restricted to the spermatogenic lineage owing to the seminiferous
tubular environment in which they reside. Once outside of this environment,
they can switch to another lineage depending on the particular niche in which
they are placed.
|
An important question is what changes in gene expression accompany the
transition from a cultured SSC to an ES-like cell? In this respect, it is
interesting to study the possible changes in the expression of those genes
that can transform a fibroblast into an ES-like cell, that is Myc,
Oct4 (Pou5f1), Sox2 and Klf4
(Takahashi and Yamanaka, 2006;
Wernig et al., 2007), in SSCs
and in the ES-like cells derived from them. Kanatsu-Shinohara et al.
(Kanatsu-Shinohara et al.,
2008) found that all four pluripotency genes are already expressed
at low levels in cultured SSCs, although no NANOG
(Kanatsu-Shinohara et al.,
2004) or SOX2 protein expression was found in these cells. In
ES-like cells, the expression of these four genes is much increased
(Table 2). In addition to these
pluripotency genes, the ES cell markers stage-specific embryonic antigen-1
(SSEA-1; FUT4) and, to a low level, Forssman antigen (GBGT1), were induced in
the ES-like cells and, as in ES cells, high levels of alkaline phosphatase
(AP) were also found (Kanatsu-Shinohara et
al., 2004). Guan et al. (Guan
et al., 2006) assayed expression patterns in SSCs cultured under
conditions that induced these cells to become ES-like cells. In this
situation, it is difficult to categorize these cells as being either SSCs or
ES-like cells as they might be in an in-between state. In these SSCs/early
ES-like cells, Oct4, Nanog and SSEA1 were expressed
(Guan et al., 2006)
(Table 2). Indeed, the level of
expression of Nanog and SSEA1 suggests that these cells were
already on their way to becoming ES-like cells. Seandel et al.
(Seandel et al., 2007) also
studied gene expression levels before and after the transition of cultured
SSCs to ES-like cells. Oct4 was present in both cell types, but
Nanog and Sox2 were strongly induced in ES-like cells,
whereas the early spermatogonial markers Stra8, Plzf
(Zbtb16), c-Ret and Dazl became inhibited
(Table 2).
|
) also carried out a microarray study and found that a great
many genes changed their expression levels during the transition from being a
cultured SSC to an ES-like cell. Among these genes, over a 100 were induced
more than 5-fold in ES-like cells as compared with cultured SSCs, and another
100 were inhibited more than 5-fold in ES-like cells. Clear differences
between the patterns of genomic imprinting are also seen between cultured SSCs
and ES-like cells. Kanatsu-Shinohara et al.
(Kanatsu-Shinohara et al.,
2004) studied the imprinting pattern of three paternally [H19,
Meg3 and Rasgrf1] and two maternally (Igf2r and
Peg10) imprinted regions in cultured SSCs and in the ES-like cells
derived from them. Cultured SSCs show a completely androgenetic (paternal)
imprinting pattern at the differentially methylated regions (DMRs) of these
genes and loci; the DMRs of H19 and Meg3 are completely
methylated and that of Igf2r is demethylated. By contrast, in the
ES-like cells, the paternally imprinted regions are methylated to different
degrees and the maternally imprinted regions (Igf2r and
Peg10) are rarely methylated. Interestingly, the methylation patterns
that are seen in the ES-like cells are not the same as those seen in proper ES
cells, as the DMRs in ES cells are generally more methylated than those in
ES-like cells, including the maternally imprinted regions. Furthermore, both
Kanatsu-Shinohara et al. and Seandel et al. reported that most of the ES-like
cells obtained had a normal karyotype and that there was no evidence of clonal
cytogenetic abnormalities
(Kanatsu-Shinohara et al.,
2004; Seandel et al.,
2007). However, recently, Takahashi et al.
(Takehashi et al., 2007) did
find some SSC-derived ES-like cells that were trisomic for chromosomes 8 or
11, which is a common chromosomal abnormality in ES cells. In conclusion, the transition from cultured SSCs to ES-like cells is accompanied by extensive changes in gene expression, during which three of the four pluripotency genes (the exception being Oct4, which is already expressed in mouse SSCs) become expressed at higher levels, along with many other genes. Furthermore, changes occur in the genomic imprinting patterns of these cells as they undergo this transition. Although the ES-like cells acquire the expression of ES cell-specific genes, the expression pattern of these genes in ES-like cells is not identical to that seen in normal ES cells, with differences evident, for example, in the expression of brachyury, Gdf3, Forssman antigen, Nog and Stra8 (see Table 2).
Differentiation of SSC-derived ES-like cells
Kanatsu-Shinohara et al.
(Kanatsu-Shinohara et al.,
2004), Guan et al. (Guan et
al., 2006) and Seandel et al.
(Seandel et al., 2007) were
all able to derive teratomas from the ES-like cells they obtained from
cultured SSCs. In these teratomas, derivatives of all three embryonic germ
layers were found. When ES-like cells were cultured using the `hanging drop'
method, embryoid bodies (EBs) formed that also gave rise to ectodermal-,
mesodermal- and endodermal-derived tissues
(Table 1).
Furthermore, Kanatsu-Shinohara et al.
(Kanatsu-Shinohara et al.,
2004) cultured the SSC-derived ES-like cells on an OP9 stromal
feeder layer that supports the differentiation of mesodermal cells, such as
hematopoietic or muscle cells. Within 10 days, different cell types were
identified, including hematopoietic cells, vascular cells and spontaneously
beating cardiomyocytes. Some ES-like cells were transferred onto
gelatin-coated dishes to promote the differentiation of neural lineages and
formed neurons or glial cells. Dopaminergic neurons were also found in these
cultures, albeit at low frequency. In addition, Baba et al.
(Baba et al., 2007) found that
ES-like cells cultured in this way have the potential to differentiate into
cardiomyocytes and endothelial cells.
Guan et al. (Guan et al.,
2006) applied the `hanging drop' method to induce ES cell
differentiation. Differentiation into mesodermal lineages (e.g. cardiac,
skeletal muscle and vascular cells) was confirmed by the expression of the
early mesoderm marker brachyury (T), as well as of lineage-specific
genes and proteins. In addition, single cardiomyocytes were isolated from
beating areas of the cultures. These cells showed sarcomeric striations when
stained for -sarcomeric actinin, sarcomeric MHC and cardiac troponin T.
Expression of the gap-junction protein connexin 43 (GJA1) in cardiac clusters
indicated that cell-to-cell contacts had been made in these cultures and that
cells were in communication with each other. Patch-clamp electrophysiological
studies of these single cardiomyocytes showed spontaneous action potentials
(Guan et al., 2007). The
differentiation of SSC-derived ES cell-like cells into vascular endothelial
and smooth muscle cells was confirmed by the expression of genes that encode
proteins specific for these cell types. In ES-like-cell-derived EB outgrowths,
cells were present that bore the characteristics of endothelial cells.
Neuroectoderm differentiation in these EBs was confirmed by the expression of
nestin, a marker for neuroepithelial precursors. Seandel et al.
(Seandel et al., 2007) also
derived EBs from SSC-derived ES-like cells and stimulated their
differentiation into endodermal, ectodermal and mesodermal lineages. This
group showed the presence of cytokeratin (KRT1)-positive cells derived from
ectoderm and of brachyury-positive or skeletal-muscle-myosin (MYH2)-positive
cells derived from mesoderm, as well as the presence of spontaneously beating
cardiomyocytes.
Kanatsu-Shinohara et al. and Seandel et al. also microinjected ES-like
cells into blastocysts to investigate whether these cells could contribute to
chimeras in vivo (Kanatsu-Shinohara et
al., 2004; Seandel et al.,
2007). Indeed, these cells contributed widely to the tissues of
the embryo. Guan et al. microinjected cultured SSCs into recipient mouse
seminiferous tubules, before these cells made the transition to becoming
ES-like cells, and found that they were still able to colonize the testis of a
recipient mouse with spermatogenic cells
(Guan et al., 2006).
Surprisingly, after microinjection in blastocysts cultured SSCs were also able
to contribute to the tissues of the embryo. These data are in conflict with
those of Kanatsu-Shinohara et al.
(Kanatsu-Shinohara et al.
2004; Kanatsu-Shinohara et
al., 2008), who were not able to show the colonization of
blastocysts by cultured SSCs. However, in the protocol of Guan et al., ES-like
colonies form relatively early during culture and so some of the SSCs
transplanted into the blastocysts might have already taken some steps towards
the ES-like state.
All three groups showed that cultured SSCs, before their transition into
becoming ES-like cells, can colonize a recipient mouse testis in the
transplantation assay. The intriguing question then is whether or not the
SSC-derived ES-like cells are still able to revert to the spermatogenic
lineage. Kanatsu-Shinohara et al. transplanted the ES-like cells into the
testes of W/W (Kit-/-) recipient mice
(Kanatsu-Shinohara et al.,
2004). These mice lack endogenous spermatogenesis because of a
deficiency of the c-KIT receptor, which is necessary for primordial germ cell
development and for the differentiation of SSCs. In all cases, teratomas
formed upon ES-like cell transplantation and no normal spermatogenesis was
initiated. Thus, although the ES-like cells can differentiate into many
lineages, it is unlikely that they can revert to becoming spermatogenic
cells.
Finally, in two studies, SSCs were placed in an environment that favors
tissue-specific differentiation. Boulanger et al.
(Boulanger et al., 2007)
injected a mixture of mouse testis cells and mammary cells into the mammary
fat pad and obtained differentiation of the SSCs into cells that have mammary
epithelial progenitor cell properties; mammary epithelial cells of SSC origin
also formed. Hu et al. (Hu et al.,
2007) cultured germ cells from 6- to 8-day-old mouse testes under
conditions that favor osteoblast differentiation [the cells were cultured in
Iscove's Modified Dulbecco's Medium (IMDM) plus dimethylsulfoxide and FGF2].
In this culture, cells appeared that stained for both collagen I and for AP
activity in the supernatant (collagen I and AP are markers of osteoblasts). AP
levels were low early on in culture and increased gradually. Thus, these two
studies suggest that SSCs can transdifferentiate into other cell lineages. As
in the mammary gland experiment, no teratomas were found. As such, this
transdifferentiation event might not proceed via the formation of ES-like
cells; if this were the case, teratomas would probably have formed.
In conclusion, the SSC-derived ES-like cells can differentiate into a great many cell lineages, either by their being directly cultured according to differentiation protocols developed for ES cells, or via the formation of EBs by these cells. In addition, some studies suggest that SSCs can transdifferentiate into non-spermatogenic cell types when placed directly into an environment that stimulates a particular differentiation pathway.
Origin of the ES-like cells
An important question that is raised by these studies is the origin of the
ES-like cells that are derived from cultured testicular cells. In order to
answer this question, Seandel et al.
(Seandel et al., 2007) used
knock-in mice bearing a GPR125-β-galactosidase fusion protein under the
control of the native Gpr125 promoter. Gpr125 encodes an
orphan adhesion type G protein-coupled receptor, and in the testis is only
expressed in spermatogonia and early spermatocytes. Subsequently, this group
found β-galactosidase staining in the testis-derived ES-like cells, as
well as in the teratomas, EBs and chimeric mice partly formed by the
transplantation of these ES-like cells into blastocysts. This clearly shows
that the ES-like cells produced are of germ cell origin. A second question
that arises is whether, in addition to the SSCs that maintain normal
spermatogenesis, there exists in the testis a residual small population of
multipotent cells that might originate from the fetal testis, from which the
ES-like colonies form in culture. This question was recently answered by
Kanatsu-Shinohara et al., who showed that in a cultured SSC line derived from
one cultured SSC, a colony of ES-like cells could arise alongside normal SSCs
(Kanatsu-Shinohara et al.,
2008). The ES-like cells that formed on this occasion could also
be propagated further as ES-like cells in an appropriate ES medium. After this
event, the SSC line was cultured for another 6 months without forming another
ES-like colony. This indicates that ES-like cells can derive anew from normal
cultured SSCs and that each SSC apparently has a small chance to convert from
being a cultured SSC to an ES-like cell. Unfortunately, as the molecular
mechanisms that bring about this transition are as yet completely unknown,
there are no clues as to how we can influence the occurrence of this chance
event. These findings indicate that the formation of ES-like cells from germ
cells does not depend on the presence of a specific, residual population of
potentially multipotent germ stem cells, other than the normal SSCs that
remain after birth. The data rather suggest that all SSCs in principle have
the potential to become multipotent again. However, in the normal seminiferous
epithelium, this capacity never becomes apparent and then only rarely under
the culture conditions that are presently used for SSCs.
|
The findings discussed above offer a number of new research tools and pose
new questions to be answered. The transition from being a stem cell that is
dedicated to a specific lineage to being a multipotent stem cell, and the
regulation of this step, are of considerable, fundamental interest. This
process can now be studied using the SSC culture system. However, the rate of
progress in this direction will depend on our finding a way to enhance the
likelihood that this transition will occur because, in the current protocols,
this transition is still a relatively rare event, as described by
Kanatsu-Shinohara et al.
(Kanatsu-Shinohara et al.,
2004; Kanatsu-Shinohara et
al., 2008) but not by the other groups; in their experiments, it
was probably a rare event too. A first step in finding ways to enhance the
chance that a cultured SSC will become an ES-like cell has been provided by
Kanatsu-Shinohara et al.
(Kanatsu-Shinohara et al.,
2008) in their comparison of gene expression patterns between SSCs
and the ES-like cells derived from them. This approach might provide clues
about how to stimulate this transition.
Another important point is whether the results obtained in mice can also be
achieved in other mammalian species, particularly in humans. If applicable to
human SSCs, these techniques could offer new opportunities in the field of
regenerative medicine. However, for these possibilities to be realised, new
differentiation protocols will have to be developed and ways found to prevent
the formation of teratomas. In addition, spermatogonial multiplication and
stem cell renewal in primates differ from those in non-primate mammals and are
not yet fully understood (de Rooij and
Russell, 2000; Ehmcke and
Schlatt, 2006). Human SSCs might be different in nature and might
be regulated in different ways, and they might also have different levels of
susceptibility to making the transition to an ES-like cell.
From the discussions above, it is clear that the method used to isolate
SSCs is not of crucial importance, and Kanatsu-Shinohara et al.
(Kanatsu-Shinohara et al.,
2004) and Seandel et al.
(Seandel et al., 2007) used
roughly the same culture protocol to generate ES-like cells, with some
differences in feeder layers and in the time taken for ES cell-like cells to
emerge (Fig. 3). The culture
protocol of Guan et al. (Guan et al.,
2006) was different in that GDNF was only added during the first
4-7 days of culture and a simple medium was then used with added serum. Taken
together, from these studies one can speculate that the time for ES-like
colonies to develop depends on the resemblance of the culture system to the
original SSC niche. It took the longest in the Seandel et al. study
(Seandel et al., 2007), in
which testicular peritubular and Sertoli cells were used as a feeder layer and
GDNF was added, and the least time in the Guan et al. method
(Guan et al., 2006), in which
no MEFS or feeder layer was provided, and GDNF was given only at the start of
the cultures. Conversely, Kanatsu-Shinohara et al.
(Kanatsu-Shinohara et al.,
2004) found that ES-like colonies failed to form when the SSCs
from neonatal mice were cultured in ES medium right from the start.
The transition from cultured SSCs to ES-like cells is accompanied by
extensive changes in gene expression. Cultured SSCs express many germ
cell-specific genes. The ES-like cells that originate from the SSCs show high
levels of expression of the pluripotency genes (Oct4, Sox2, Myc and
Klf4), as well as of their encoded proteins. Furthermore, germ
cell-specific genes are downregulated. The important question is how the
expression of the pluripotency genes is regulated. As noted by
Kanatsu-Shinohara et al.
(Kanatsu-Shinohara et al.,
2008), as the pluripotency genes are already expressed in cultured
SSCs, one has to assume that in these cells there is a mechanism through which
the expression of these genes is kept at a low level. Furthermore, upon
transition into ES-like cells, the expression of the germ cell-specific genes
has to be shut down.
The three studies in which SSCs were cultured and then ES-like cells
appeared all demonstrate the multipotent character of the ES-like cells, which
can give rise to derivatives of all three germ layers, teratomas, EBs and to
chimaeras (upon injection into blastocysts). Using ES cell differentiation
protocols or via EB formation, SSCs have also been differentiated into a
myriad of cell types. Interestingly, differences also exist in the gene
expression patterns and epigenetic marks of SSC-derived ES-like cells as
compared with those of ES cells and induced pluripotent cells (iPS)
(Kanatsu-Shinohara et al.,
2004; Kanatsu-Shinohara et
al., 2008). It will be of fundamental importance to study the
differences in the differentiation patterns between these cell types because
particular ES-like cell types might be more suitable for producing a
particular differentiated cell type.
In two studies, a transition of SSCs into other cell lineages was reported
without being preceded by culture and by the transition of SSCs into ES-like
cells (Boulanger et al., 2007;
Hu et al., 2007). These
findings will have to be confirmed and studied in further detail to establish
more precisely how the transition of SSCs into cells of another lineage takes
place. In itself, for regenerative medicine purposes, it would be of great
advantage if SSCs could be made to differentiate into other cell lineages
without becoming multipotent ES-like cells first, as these ES-like cells have
the potential to form teratomas.
Remarkably, so far all the data published on the plasticity of SSCs have been obtained in mice. We still have to wait to see whether the conclusions drawn for mouse SSCs also hold true for other mammals, especially humans. Needless to say, the implications will be enormous if SSCs from humans could be used to produce ES-like cells that could be made to differentiate into a tissue a patient needs. There have been statements that this has already been achieved in humans (e.g. http://goliath.ecnext.com/coms2/summary_0199-5356645_ITM), but, so far, these have not been substantiated by publications in peer-reviewed international journals.
ACKNOWLEDGMENTS
The authors are supported by the European Science Foundation (ESF) program EuroSTELLS, through Zon-Mw 910-20-032.
REFERENCES
Baba, S., Heike, T., Umeda, K., Iwasa, T., Kaichi, S., Hiraumi,
Y., Doi, H., Yoshimoto, M., Kanatsu-Shinohara, M., Shinohara, T. et al.
(2007). Generation of cardiac and endothelial cells from neonatal
mouse testis-derived multipotent germline stem cells. Stem
Cells 25,1375
-1383.
Boulanger, C. A., Mack, D. L., Booth, B. W. and Smith, G. H.
(2007). Interaction with the mammary microenvironment redirects
spermatogenic cell fate in vivo. Proc. Natl. Acad. Sci.
USA 104,3871
-3876.
Brinster, R. L. and Avarbock, M. R. (1994).
Germline transmission of donor haplotype following spermatogonial
transplantation. Proc. Natl. Acad. Sci. USA
91,11303
-11307.
Brinster, R. L. and Zimmermann, J. W. (1994).
Spermatogenesis following male germ-cell transplantation. Proc.
Natl. Acad. Sci. USA 91,11298
-11302.
Buageaw, A., Sukhwani, M., Ben-Yehudah, A., Ehmcke, J., Rawe, V.
Y., Pholpramool, C., Orwig, K. E. and Schlatt, S. (2005).
GDNF family receptor alpha1 phenotype of spermatogonial stem cells in immature
mouse testes. Biol. Reprod.
73,1011
-1016.
Creemers, L. B., Meng, X., Den Ouden, K., Van Pelt, A. M.,
Izadyar, F., Santoro, M., Sariola, H. and de Rooij, D. G.
(2002). Transplantation of germ cells from glial cell
line-derived neurotrophic factor-overexpressing mice to host testes depleted
of endogenous spermatogenesis by fractionated irradiation. Biol.
Reprod. 66,1579
-1584.
Cyranoski, D. (2006). Stem cells from testes: could it work? Nature 440,586 -587.[CrossRef][Medline]
de Rooij, D. G. (2001). Proliferation and differentiation of spermatogonial stem cells. Reproduction 121,347 -354.[Abstract]
de Rooij, D. G. (2006). Rapid expansion of the
spermatogonial stem cell tool box. Proc. Natl. Acad. Sci.
USA 103,7939
-7940.
de Rooij, D. G. and Russell, L. D. (2000). All you wanted to know about spermatogonia but were afraid to ask. J. Androl. 21,776 -798.[Medline]
Ehmcke, J. and Schlatt, S. (2006). A revised
model for spermatogonial expansion in man: lessons from non-human primates.
Reproduction 132,673
-680.
Gilbert, S. F. (2003). Developmental Biology, 7th edn. Sunderland, MA: Sinauer Associates.
Guan, K., Nayernia, K., Maier, L. S., Wagner, S., Dressel, R., Lee, J. H., Nolte, J., Wolf, F., Li, M., Engel, W. et al. (2006). Pluripotency of spermatogonial stem cells from adult mouse testis. Nature 440,1199 -1203.[CrossRef][Medline]
Guan, K., Wagner, S., Unsold, B., Maier, L. S., Kaiser, D.,
Hemmerlein, B., Nayernia, K., Engel, W. and Hasenfuss, G.
(2007). Generation of functional cardiomyocytes from adult mouse
spermatogonial stem cells. Circ. Res.
100,1615
-1625.
Hofmann, M. C., Braydich-Stolle, L. and Dym, M. (2005). Isolation of male germ-line stem cells; influence of GDNF. Dev. Biol. 279,114 -124.[CrossRef][Medline]
Hu, H. M., Xu, F. C., Li, W. and Wu, S. H. (2007). Biological characteristics of spermatogonial stem cells cultured in conditions for fibroblasts. J. Clin. Rehab. Tiss. Engin. Res. 11,6611 -6614.
Kanatsu-Shinohara, M. and Shinohara, T. (2006). The germ of pluripotency. Nat. Biotechnol. 24,663 -664.[CrossRef][Medline]
Kanatsu-Shinohara, M., Ogonuki, N., Inoue, K., Miki, H., Ogura,
A., Toyokuni, S. and Shinohara, T. (2003). Long-term
proliferation in culture and germline transmission of mouse male germline stem
cells. Biol. Reprod. 69,612
-616.
Kanatsu-Shinohara, M., Inoue, K., Lee, J., Yoshimoto, M., Ogonuki, N., Miki, H., Baba, S., Kato, T., Kazuki, Y., Toyokuni, S. et al. (2004). Generation of pluripotent stem cells from neonatal mouse testis. Cell 119,1001 -1012.[CrossRef][Medline]
Kanatsu-Shinohara, M., Ogonuki, N., Iwano, T., Lee, J., Kazuki,
Y., Inoue, K., Miki, H., Takehashi, M., Toyokuni, S., Shinkai, Y. et al.
(2005). Genetic and epigenetic properties of mouse male germline
stem cells during long-term culture. Development
132,4155
-4163.
Kanatsu-Shinohara, M., Lee, J., Inoue, K., Ogonuki, N., Miki,
H., Toyokuni, S., Ikawa, M., Nakamura, T., Ogura, A. and Shinohara, T.
(2008). Pluripotency of a single spermatogonial stem cell in
mice. Biol. Reprod. 78,681
-687.
Kubota, H. and Brinster, R. L. (2006). Technology insight: In vitro culture of spermatogonial stem cells and their potential therapeutic uses. Nat. Clin. Pract. Endocrinol. Metab. 2,99 -108.[CrossRef][Medline]
Meng, X., Lindahl, M., Hyvonen, M. E., Parvinen, M., de Rooij,
D. G., Hess, M. W., Raatikainen-Ahokas, A., Sainio, K., Rauvala, H., Lakso, M.
et al. (2000). Regulation of cell fate decision of
undifferentiated spermatogonia by GDNF. Science
287,1489
-1493.
Nakagawa, T., Nabeshima, Y. and Yoshida, S. (2007). Functional identification of the actual and potential stem cell compartments in mouse spermatogenesis. Dev. Cell. 12,195 -206.[CrossRef][Medline]
Nayernia, K. (2007). Stem cells derived from testis show promise for treating a wide variety of medical conditions. Cell Res. 17,895 -897.[CrossRef][Medline]
Nayernia, K. (2008). Potency of germ cells and role in regenerative medicine. J. Anat. 212, 75-76.
Seandel, M., James, D., Shmelkov, S. V., Falciatori, I., Kim, J., Chavala, S., Scherr, D. S., Zhang, F., Torres, R., Gale, N. W. et al. (2007). Generation of functional multipotent adult stem cells from GPR125+ germline progenitors. Nature 449,346 -350.[CrossRef][Medline]
Takahashi, K. and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126,663 -676.[CrossRef][Medline]
Takehashi, M., Kanatsu-Shinohara, M., Miki, H., Lee, J., Kazuki, Y., Inoue, K., Ogonuki, N., Toyokuni, S., Oshimura, M., Ogura, A. et al. (2007). Production of knockout mice by gene targeting in multipotent germline stem cells. Dev. Biol. 312,344 -352.[CrossRef][Medline]
Tegelenbosch, R. A. and de Rooij, D. G. (1993). A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H/101 F1 hybrid mouse. Mutat. Res. 290,193 -200.[CrossRef][Medline]
Wernig, M., Meissner, A., Foreman, R., Brambrink, T., Ku, M., Hochedlinger, K., Bernstein, B. E. and Jaenisch, R. (2007). In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448,318 -324.[CrossRef][Medline]
Yeh, J. R., Zhang, X. and Nagano, M. C. (2007).
Establishment of a short-term in vitro assay for mouse spermatogonial stem
cells. Biol. Reprod. 77,897
-904.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Takashima, M. Takehashi, J. Lee, S. Chuma, M. Okano, K. Hata, I. Suetake, N. Nakatsuji, H. Miyoshi, S. Tajima, et al. Abnormal DNA Methyltransferase Expression in Mouse Germline Stem Cells Results in Spermatogenic Defects Biol Reprod, July 1, 2009; 81(1): 155 - 164. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Gromley, M. L. Churchman, F. Zindy, and C. J. Sherr Transient expression of the Arf tumor suppressor during male germ cell and eye development in Arf-Cre reporter mice PNAS, April 14, 2009; 106(15): 6285 - 6290. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Lee, M. Kanatsu-Shinohara, N. Ogonuki, H. Miki, K. Inoue, T. Morimoto, H. Morimoto, A. Ogura, and T. Shinohara Heritable Imprinting Defect Caused by Epigenetic Abnormalities in Mouse Spermatogonial Stem Cells Biol Reprod, March 1, 2009; 80(3): 518 - 527. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
