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Human Reproduction Update, Vol.15, No.3 pp. 379– 390, 2009
Advanced Access publication on January 28, 2009 doi:10.1093/humupd/dmp001
Differentiation of germ cells
and gametes from stem cells
A.I. Marques-Mari 1, O. Lacham-Kaplan 2, J.V. Medrano 1,
A. Pellicer 3, and C. Simón 1,3,4
1
Valencia Stem Cell Bank, Centro de Investigación Prı́ncipe Felipe (CIPF), 46012 Valencia, Spain 2Monash Immunology and Stem Cell
Laboratories (MISCL), Monash University, 3800 Clayton, Australia 3Fundacion Instituto Valenciano de Infertilidad, Valencia University,
C/Guadassuar, 1, E-46015 Valencia, Spain
4
Correspondence address. E-mail: [email protected]
table of contents
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Introduction
Methods
Specification of germ cells in mammals
Epigenetic modifications in the germline
In vitro differentiation of gametes from SC
Demanding biological descent: how to obtain patient-specific gametes
Applications and future prospects
background: Advances in stem cell research have opened new perspectives for regenerative and reproductive medicine. Stem cells
(SC) can differentiate under appropriate in vitro and in vivo conditions into different cell types. Several groups have reported their ability to
differentiate SCs into germline cells, and some of them have been successful in obtaining male and female gamete-like cells by using different
methodologies.
methods: This review summarizes the current knowledge in this field and emphasizes significant embryological, genetic and epigenetic
aspects of germ cells and gametes in vitro differentiation in humans and other species, highlighting major obstacles that need to be overcome
for successful gametogenesis in culture: studies reporting development of germ cell-like cells from murine and human embryonic (ESC) and
somatic SCs are critically reviewed.
results: Published studies indicate that germ cells can be consistently differentiated from mouse and human ESC. However, further
differentiation of germ cells through gametogenesis still has important genetic and epigenetic obstacles to be efficient.
conclusions: Differentiation of germ cells from SCs has the potential of becoming a future source of gametes for research use,
although further investigation is needed to understand and develop the appropriate niches and culture conditions. Additionally, if genetic
and epigenetic methodological limitations could be solved, therapeutic opportunities could be also considered.
Key words: differentiation / gametes / germ cells / embryonic stem cells / reproductive medicine
Introduction
Stem cells (SC) are undifferentiated cells that have the potential to
self-replicate and give rise to specialised cells. These include cells
lost from normal turnover or from a disease in specific organs and
tissues, reflecting on their possible use in future therapies.
SCs can be obtained from the embryo at cleavage or blastocyst
stages (embryonic stem cells, ESC), but also from extra-embryonic
tissues such as the umbilical cord blood obtained at birth (McGuckin
et al., 2005), the placenta (Miki et al., 2005) and the amniotic fluid (De
Coppi et al., 2007). SC can also be obtained in adult mammals from
specific niches. These somatic SCs can be found in a wide range of
tissues including bone marrow (Pittenger et al., 1999), blood
(Goodman and Hodgson, 1962; Barnes and Loutit, 1967), fat (Zuk
et al., 2002), skin (Toma et al., 2001; Alonso y Fuchs, 2003) and
also the testis (Guan et al., 2006).
ESCs exposed to appropriate and specific conditions differentiate
into cell types of all three germ layers (endoderm, ectoderm and
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380
mesoderm) and also into germline cells. The latter had raised speculations that ESCs may have a potential role in reproductive medicine.
According to the World Health Organisation (WHO), infertility is
considered a disease affecting millions of people in Europe, where
prevalence is 14% of couples in reproductive age. Based on the
2005 National Survey on Family Growth American report, there
was a 20% increase in American couples experiencing impaired
fecundity between 1995 and 2002. This may be related to a tendency
to delay motherhood to the third decade of life due to professional
and social reasons. As a consequence, oocyte quality in females is
reduced (Howe, 1985; Spira, 1988; Maroulis, 1997; Ziebe et al.,
2001).
The already short reproductive period in women can be even shortened if oocytes or granulosa cells are damaged following cancer treatments; consequently, assisted fertilization is required.
In males, sperm are produced continuously during the adult life,
hence, spermatogenesis may be re-established through progenitor
germ SC within the testis. In the case of radiation, the damage is
dose dependent, leading from transient to permanent infertility in
men, and consequently, to the necessity of assisted fertilization treatments (Kinsella, 1989; Lampe et al., 1997). The option of storing
mature sperm prior to treatment is a common practice, but this possibility does not exist for pre-pubertal cancer patients. For these
patients, transplantation of spermatogonial stem cells (SSCs) obtained
before treatment is the only possible strategy to restore fertility,
although with the risk of reseeding cancer cells back to them.
Failure of patients to obtain viable embryos and pregnancies
through assisted reproductive techniques (ART) due to the reduced
quality of their gametes leaves them with the option of receiving
donated oocytes and sperm. From ART cycles performed in 2000
in 49 countries worldwide, 32.3% of the procedures involved
oocyte donation (Adamson et al., 2006). In the USA alone in 2004,
12.5% of ART cycles were carried out with donor oocytes (ART
success rates 2004. US Department of Health and Human Services).
Data published by the European Society of Human Reproduction
and Embryology (ESHRE), showed that the proportion of ART
cycles with oocyte donation increased by approximately 20% from
2003 to 2004 in Europe (Andersen et al., 2007, 2008). Although pregnancy rates with these procedures exceed 50%, oocyte donation
always raises ethical, legal and personal concerns. In addition, patients
value biological parenthood and desire their own biological
descendant.
Methods
The development and improvement of the research on ESC-derived
gametes has gained attention from the reproductive biology field in
recent years. Several studies reported that murine ESCs can develop
into germ cell precursor cells in vitro (Hübner et al., 2003; Toyooka
et al., 2003; Geijsen et al., 2004; Lacham-Kaplan et al., 2006; Nayernia
et al., 2006b; Novak et al., 2006; Qing et al., 2007). Also human ESCs
(hESCs) show spontaneous and induced differentiation into cells with
markers of germ cells (Clark et al., 2004; Kee et al., 2006; Chen et al.,
2007).
The present critical review will outline progress in research into ESCderived gametes in mice and humans in recent years, focusing on the
potential benefits that this development may hold for reproductive therapies. In addition, this paper will outline possibilities to improve the
Marques-Mari et al.
outcome of these procedures to result in viable gametes, which will be
accepted for clinical use. For this purpose, the more knowledge about
the processes of gametogenesis in vivo the better to replicate them properly to obtain viable gametes in vitro.
Specification of germ cells
in mammals
Germ cells are the biological route for genetic transmission from one
generation to the next. These cells constitute a very different cell
population from somatic cells, with unique characteristics, and
display a haploid chromosomal number after a delicate process of
meiosis. But, how do these cells acquire a different fate than the
other cells of the individual? Can we induce this same behaviour in
undifferentiated SC in vitro?
In humans, and mammals in general, germ cells are derived from a
specific pluripotent population known as primordial germ cells
(PGCs), which segregate early in embryogenesis and become progenitors of adult gametes. The first PGCs in humans are located in the yolk
sac outside the embryo at 2 –3 weeks of development (Witschi,
1948). In mice, they are originated from a founder population of
cells located at the base of the allantois on Day E7.25 (Ginsburg
et al., 1990) in response to inductive signals emanating from the adjacent extra-embryonic ectoderm, including bone morphogenetic proteins (BMPs), particularly BMP-4 and -8 (Lawson et al., 1999; Ying
et al., 2000, 2001; Fujiwara et al., 2001; Saitou et al., 2002). In addition
to PGCs, the pluripotent epiblast cells also give rise to the three
embryonic lineages: the mesoderm, ectoderm and endoderm. While
these three lineages continue their differentiation into other tissues
in the body, PGCs retain pluripotency as identified by the expression
of specific markers such as Oct4 and Nanog (Scholer et al., 1990;
Pesce et al., 1998; Mitsui et al., 2003) and high alkaline phosphatase
activity (Chiquoine et al., 1954, Ginsburg et al., 1990) (Fig. 1).
Mouse and human PGCs migrate in the developing embryo towards
the gonadal ridge, proliferate and differentiate into gonocytes, the
primitive germ cells. In human, this migration takes place between
weeks 4 –6 (Witschi, 1948; Fujimoto et al., 1977; Goto et al., 1999)
and in mice between days E8.5–E12.5 (Wylie et al., 1986).
The differentiation of gonads into a primitive ovary or testis
coincides with time of arrival of the PGCs to the genital ridge, creating
the proper microenvironment or niche necessary for sex determination (Byskov, 1986).
In males, gonocytes arrest in G0 and stay mitotically quiescent until
birth (McLaren, 1984; Goto et al., 1999). At birth, quiescent cells are
triggered to enter mitotic cell cycles and differentiate into spermatogonia that undergo multiple cell divisions and differentiate into spermatocytes. These cells continue their meiosis into haploid spermatids,
which proceed into mature sperm. A small population of SSCs
(capable of renewing themselves or producing spermatogonia for
futher differentiation) remains in the testis through adulthood (Bellve
et al., 1977). These cells continuously enter the meiotic cell pool to
result in haploid cells. Similar germ stem cells (GSCs, multipotent
SCs derived from PGCs that generate sperm or oocytes) are not
thought to be present in the adult ovary although controversial
studies by Johnson and Bukovsky have challenged this dogma
(Johnson et al., 2004, 2005; Bukovsky et al., 2005). These researchers
381
Germ cells from stem cells
Epigenetic modifications
in the germline
Figure 1 The germline development: events and molecular keys.
During in vivo embryonic development, as a result of BMP4 stimulation, as well as BMP2 and BMP8b, a subset of epiblast cells begins
to express the Fragilis and Blimp1 genes and become PGCs which
migrate into the embryo towards the gonadal ridge and proliferate.
During migration these cells express other germ cell-related
markers as Stella and c-kit and initiate colonization of the gonadal
ridge. At this time, PGCs express the premeiotic markers Dazl and
Vasa. The proteins SCP1, SCP2, and SCP3 are specific of meiotic
germ cells, as well as the meiosis gene Dmc1. The female germ
cells enter meiosis and arrest in prophase I, the male germ cells
arrest in mitosis and do not enter meiosis until after birth. The postmeiotic markers GDF9 and TEKT1 are specific of more mature
haploid gametes. Dotted lines refer to postnatal development.
have observed that adult mouse and human ovaries possess mitotically
active GSCs, allowing continued renewal of the follicle pool in adult
ovaries.
In females, PGCs initiate meiosis at the same time in development
that male germ cells enter mitotic arrest. This occurs at the beginning
of week 12 of human gestation (Gondos et al., 1986) and in mice on
day E13.5 (Monk and McLaren, 1981). PGCs in the female embryo
do not complete meiosis, and are arrested at prophase I of the
first meiotic cycle. Progress in meiosis occurs post-natally just prior
to ovulation followed by another arrest in meiosis II, which is completed just after fertilization (McLaren, 1995; Goto et al., 1999)
(Fig. 1).
Genomic imprinting is a DNA modification pattern which is common
and unique to all cells of an individual. This imprinting can be found at
both genomic areas without a specific function and areas where the
expression of specific genes is controlled. Thereby imprinting can
control the proper expression levels of genes required for normal
embryonic development and cell function, without changing the
genome itself. In an organism the imprinting pattern is partially inherited from both paternal and maternal genomes. After fertilization the
imprinting marks and epi-mutations are erased by the epigenetic
machinery of the zygote, and then re-established in a new and
unique pattern depending on the sex of the new individual.
These epigenetic marks, which define the imprinting, include modifications in DNA and histones, especially methylation, acetylation,
phosphorylation and ubiquinitation, DNA methylation being the best
characterized of these mechanisms, which has been shown to have
essential functions in the germline and the embryo development.
These chromatin modifications provide a mechanism for the adequate
expression and repression of genes and hence for their temporal or
permanent inactivation (Razin and Cedar, 1991; Yeivin and Razin,
1993; Wolffe et al., 1998).
Epigenetic modifications are sequentially established and erased in
the germinal lineage. PGCs undergo DNA demethylation of the
imprinted loci as soon as they reach the gonadal ridge and the parental
patterns are erased (Monk, 1987; Brandeis et al., 1993; Lee et al.,
2002; Yamazaki et al., 2003). Re-establishment of different imprints
in germ cells of both sexes with new patterns according to the
gender of the new individual is initiated during male and female
gamete differentiation. During spermatogenesis, de novo methylation
occurs before the onset of meiosis, while in oogenesis it occurs
after the onset of meiosis (Kafri et al., 1992; Brandeis et al., 1993;
Kono et al., 1996; Davis et al., 2000; Obata and Kono, 2002)
(Fig. 2). For the zygote to acquire totipotency extensive epigenetic
reprogramming occurs. Shortly after fertilization the paternal
genome undergoes rapid demethylation, while the maternal genome
is slowly demethylated during the first cleavages (Reik and Walter,
2001; Santos et al., 2002). Then, around the time of implantation of
the blastocyst there is a wave of de novo methylation to establish
an individual specific pattern (Reik et al., 2001; Hajkova et al., 2002).
Parthenogenesis, a mechanism by which an oocyte is activated
without fertilization by a spermatozoid, has been described in some
studies after differentiation of oocyte-like cells from SC in culture
(Hübner et al., 2003; Dyce et al., 2006). However, the formed
embryos arrested in early stages of the process with no further development. It is likely that the levels of imprinted genes are unbalanced in
these embryos, suggesting that some imprints are not correctly reprogrammed in the differentiated gametes.
Factors that mediate epigenetic resetting are not known, but they
must be present in early embryos and pluripotent SC. The pluripotent
cells of the inner cell mass (ICM) and the PGCs share a common
feature: expression of the homeobox gene Nanog (Mitsui et al.,
2003; Yamaguchi et al., 2005). Nanog is responsible for the maintenance of pluripotency in SC, and loss of its expression leads to cell
differentiation (Mitsui et al., 2003; Wang et al., 2003). During mice
382
Marques-Mari et al.
Figure 2 Genetic imprinting in the germinal lineage. After fertilization the parental epigenetic pattern is erased to recover totipotency. Then, the
sequence of imprinting is modified during early embryo development; there is a demethylation process in the PGCs followed by a methylation recovery
in more mature gametes depending on the sex. There is no evidence that the imprinting patterns are being properly established during in vitro differentiation of gametes. Data from the different studies developed to date suggest that the generated germ cells present imprinting abnormalities.
embryonic development Nanog is expressed in preimplantation
embryos (morula stage and ICM of blastocysts) and in migrating and
gonadal PGCs from Day E7.75 –E12.25 (Mitsui et al., 2003; Yamaguchi
et al., 2005). Nanog expression is down-regulated in female germ cells
with the onset of meiosis at E13.5–E14.5, and in male germ cells after
mitotic arrest in E14.5 embryos (Yamaguchi et al., 2005).
A recent study by Chambers et al. (2007) shows that gonadal ridges
of chimaeras resulting from aggregation of Nanog null ESCs with wildtype morulae lack PGCs beyond E11.5, suggesting that Nanog is
required for germline development and maturation, although, the
defect is rescued by repair of the mutant allele. The authors
propose that the primary function of Nanog may be to establish
ICM and PGC states in vivo and to promote nuclear reprogramming
into a state with minimal epigenetic modifications and consequently
with a broad developmental potential. However, further work needs
to be performed to show that this is indeed the case.
In vitro differentiation
of gametes from SC
In vitro development of germ cells from SC to obtain mature, haploid
male and female gametes with the capacity to participate in normal
embryo and fetal development has been attempted for the last 5
years. To date, some studies have been published involving differentiation of mouse and human SC, either embryonic or somatic, into
germ cells and both male and female presumptive gametes. Nevertheless, the accurate functionality of these structures still needs to be
demonstrated (Table I).
GSCs appear spontaneously in ESC cultures indicating that, similar
to their ability to give rise to many other lineages following the
removal of pluripotency maintaining factors, such as feeder cells and
leukaemia-inhibiting factor (LIF) (in the mouse), ESCs are also reprogrammed to go through the germline lineage (Hübner et al., 2003;
Toyooka et al., 2003; Clark et al., 2004; Geijsen et al., 2004; LachamKaplan et al., 2006; Chen et al., 2007; Qing et al., 2007). Exogenous
addition of factors such as BMP4 and retinoic acid (RA) (Toyooka
et al., 2003; Geijsen et al., 2004, Nayernia et al., 2006b) improves
recruitment of GSCs and enhances their maintenance in vitro, but
does not support their progress through meiosis.
One of the major difficulties in the in vitro differentiation of germ cells
is the lack of appropriate molecular markers for the characterization of
the differentiated germ cells to distinguish them from the somatic cells.
Most of the markers used for PGCs are present in ESCs as well: Fragilis,
Stella, DAZL, c-Kit (Clark et al., 2004). Therefore, it is very difficult to
distinguish early germ cells from undifferentiated ESCs. Meiotic and
post-meiotic markers are more reliable markers, but it has been
demonstrated that the progression through the meiotic process is still
a challenge in the in vitro differentiation of gametes (Table II). The transfection of ESC lines with marked or fluorescent proteins linked to
specific gene promoters (genes implicated in pluripotency or germ
cell line development) enables the visualization of the cells in which
the specific gene of interest is expressed during the differentiation
process. However, the use of transfected lines disqualifies the putative
gametes obtained for their application in clinical procedures.
In vitro germ cells differentiation from ESCs
Basically, two different methods, with some variants, have been used
for germ cells differentiation from human and murine ESCs. One of
383
Germ cells from stem cells
Table I Summary of the included studies on germ cell differentiation from stem cells
Publication
Origin
Strategy for differentiation
Cell type obtained
Offspring
.............................................................................................................................................................................................
Germ cell differentiation from ESCs
Hübner et al. (2003)
mESC—XX, XY
SD in adherent cultures
Oocyte-like cells
No, PB
Toyooka et al. (2003)
mESC—XY
EBs formationþAC with growth factors
PGCs in vitro, sperm in vivo when
transplanted
NT
Geijsen et al. (2004)
mESC—XY
RA addition to EBs-derived cells
Spermatids
No, OF
Clark et al. (2004)
hESC—XX, XY
SD through EBs formation
Oocyte-like cells (although TEKT1
expression was found)
NT
Lacham-Kaplan et al.
(2006)
mESC—XY
EBs with conditioned media from testicular cell culture
Immature oocyte-like cells
NT
Novak et al. (2006)
mESC—XY
SD in adherent cultures and through EBs formation
Ovarian follicles
NT
Nayernia et al.
(2006a)
mESC—XY
RA addition to SSC lines-derived EBs
Sperm
Yes
Kee et al. 2006
hESC—XX
SD through EBsþgrowth factors addition
Oocyte-like cells
NT
Qing et al. (2007)
mESC—XY
EBs co-cultured with ovarian granulose cells
Oocyte-like cells
NT
Chen et al. (2007)
hESC—XX
SD in adherent cultures and through EBs formation
Oocyte-like cells
No, FD
Germ cell differentiation from somatic SCs
Johnson et al. (2004)
GSC in OSE
Histological analysis, Mvh and SCP3 IHC, BrdU-Mvh
coexpression, ovarian fragments graft
Formation of ovarian follicles after graft
NT
Johnson et al. (2005)
GSC in female BM
and blood
Analysis of germ cells markers, BM and blood
transplantations
Formation of ovarian follicles after graft
NT
Bukovsky et al. (2005)
GSC in OSE
Culture medium with estrogenic stimuli
Morphological and IHC analysis
Oocyte-like cells and granulosa-like
cells
NT
Dyce et al. (2006)
Fetal porcine skin
Medium with follicular fluid, addition of gonadotrophins
Oocytes markers and steroids production
Oocyte-like cells and blastocyst-like
structures
PB
Nayernia et al.
(2006b)
SCs in mouse BM
BM cells in adherent culture, RA addition,
transplantation into testis
Analysis of PGC, SSC and spermatogonia markers
Analysis after graft
Spermatogonia-like cells
NT
Drusenheimer et al.
(2007)
SCs in human BM
Expression of early germ cell markers and male germ
cells specific markers
Male germ cells
NT
Different strategies have been used to differentiate germ cells from embryonic stem cells, most of them based in spontaneous differentiation through EBs formation with or without
combining addition of soluble factors to the culture media. Several studies have described the possibility of post-natal oocyte generation in females, not without controversy. The source of
these cells (ovarian epithelium, bone marrow or fetal skin) still needs to be confirmed in most of them. AC, aggregation cultures with cells producing growth factors; BM, bone marrow;
EBs, embryoid bodies; FD, follicles degeneration; hESC, human embryonic stem cells; IHC, immunohistochemistry; GSC, germ stem cells; mESC, mouse embryonic stem cells; NT, not
tested; OF, oocyte fertilization; OSE, ovarian surface epithelium; PB, parthenogenic blastocyst; PGCs, primordial germ cells; RA, retinoic acid; SD, spontaneous differentiation; SSC,
spermatogonial stem cells.
them consists in spontaneous differentiation in adherent culture after
removing factors that promote pluripotency as feeders and basic fibroblast growth factor (bFGF) or LIF (Hübner et al., 2003; Nayernia et al.,
2006b; Novak et al., 2006; Chen et al., 2007), whereas the second
one implicates the formation of three-dimensional structures known
as embryoid bodies (EBs, Toyooka et al., 2003; Clark et al., 2004;
Geijsen et al., 2004; Lacham-Kaplan et al., 2006; Chen et al., 2007;
Qing et al., 2007).
Using the first method, Hübner et al. (2003) differentiated a mESC
transgenic line with specific green fluorescent protein (GFP)-tagged
Oct4 expression in germ cells and reported the obtaining of floating
structures mimicking ovarian follicles, which extruded a central cell
after gonadotrophin stimulation. PCR analysis showed the expression
of oocyte-specific markers as ZP-2, ZP-3 and FIGa but not ZP-1,
maybe this is the reason why the zona pellucida was very fragile.
Although the presence of the meiotic protein SCP3 indicated entry
of the putative oocytes in the meiotic process, Novak et al. (2006)
did not detect other meiotic proteins as SCP1 and SCP2 or Rec8
when they replicated the same experiment. Furthermore, no evidence
of chromosomal synapsis formation was detected. Then, although the
meiotic process is initiated in some cells, the meiotic programme fails
to progress correctly in vitro. Some of these structures were spontaneously activated leading to the formation of parthenogenic
embryos which arrested and degenerated in early stages of
development.
Toyooka et al. (2003) and Geijsen et al., (2004) obtained male germ
cells from mouse ESCs through EBs formation combined with the use
of knock-in cell lines with markers associated with pluripotency or
germline characteristic genes. The EBs are three-dimensional structures formed by the aggregation of undifferentiated ESCs, in which
different cell types from the three embryonic germ layers can be
formed, but also cells of the germ lineage.
384
Marques-Mari et al.
Table II Molecular markers expressed during the different development and maturation stages of germ cells and
gametes
Gene
Protein function
ESCs
PGCs
Migrating/premeiotic
Meiosis
Post-meiotic
.............................................................................................................................................................................................
Oct4
TF associated with pluripotency
þ
þ
þ
þ
ND
Nanog
TF associated with pluripotency
þ
þ
þ
þ
ND
Blimp1
First indicator of germ cell fate
ND
þ
ND
ND
ND
Fragilis
Early indicador of germ cell fate
þ
þ
þ
ND
ND
Stella
Early germline gene
þ
þ
þ
þ
þ
c-Kit
Migration and survival of PGCs
þ
þ
þ
2
2
Dazl
Differentiation and maturation of PGCs
þ
þ
þ
þ
þ
VASA/Mvh
Premeiotic germ cell-specific marker
2
2
þ
þ
þ
SCP1
Meiotic-specific markers
2
2
2
þ
þ
SCP2
Elements of the synaptonemal complex
2
2
2
þ
þ
2
2
2
þ
þ
SCP3
Dmc1
Meiotic-specific marker
2
2
2
þ
þ
FIGa
Oocyte marker
2
2
2
þ
þ
GDF9
Oocyte marker
2
2
2
þ
þ
ZP12ZP3
Zona pellucida markers
2
2
2
þ
þ
Tekt-1
Spermatocyte marker
2
2
2
þ
þ
Acrosin
Male gametes marker
2
2
2
þ
þ
Haprin
Haploid marker
2
2
2
þ
þ
TP1
Sperm marker
2
2
2
þ
þ
ESCs, embryonic stem cells; ND, not determined; PGCs, primordial germ cells (in vivo); TF, transcription factor.
Toyooka et al. (2003) formed aggregates of selected cells positive
for the germ cell marker VASA from the EBs with dissociated male
embryonic gonads, and transplanted them into the testis of male
mice to test the developmental potential of the differentiated cells.
Although marked spermatozoids were detected in the seminiferous
tubules of these animals, no further analysis of the functionality of
these sperm was performed.
Geijsen et al. (2004) analysed the imprinting status of the obtained
cells and reported that these cells showed erasure of the methylation
pattern in some imprinted loci, suggesting that PGCs obtained in vitro
display some properties of PGCs developing in vivo. Although some
haploid cells were found, the results suggested that meiosis was
highly inefficient in the EBs environment. Finally, the authors investigated the biological function of the haploid cells obtained via their
capacity to fertilize oocytes by intracytoplasmic injection. About
20% of the fertilized oocytes progressed to blastocyst stage, but it
was not tested if the embryos were capable to develop normally
with uterine transfer.
Although the number of imprinted genes represents a minority of
the whole human genome (2 –5%), most of them seem to have critical
effects on fetal growth and development. A correct imprinting is mandatory in gametes and the fertilized zygote for proper development
and cell function. Imprinting alterations are implicated in abnormal
fetal development and several diseases such as Prader-Willi, Angelmann, Beckwith-Wiedemann, Turner and Russell-Silver (Cassidy
et al., 2000; Tycko and Morison, 2002).
The imprinting regulation of the germline must be considered when
differentiation of gametes from ESCs is attempted. It is possible that
generation of gametes in vitro from ESCs results in suboptimal epigenetic programming inducing alterations in embryonic growth and
development. The only group that reported on the birth of live offspring from ESC-derived gametes (Nayernia et al., 2006b) analysed
the methylation status of several imprinted regions in the differentiated
cells and found that methylation imprints were not correctly profiled in
pups. This study is the first that demonstrates abnormalities in imprinting related to in vitro-derived germ cells and the progeny generated
using them. The pups showed growth abnormalities (some of them
were larger and others smaller), and most died during the first
months of life, emphasizing the crucial concept that imprinting status
of the derived gametes from stem cells must be carefully studied
(Fig. 2).
Taking a step back, scientists need to reconsider their strategies
when related to in vitro gametogenesis by looking at the in vivo processes. Considerations need to include stage-specific events of
imprint erasure and re-establishment, global demethylation and
re-methylation and meiosis, leading to completion of oogenesis and
spermatogenesis in relation to the gonad environment as a whole.
Meiosis is a specific germ cell skill. During meiosis, a single DNA
replication step is followed by two consecutive cell divisions, generating haploid gametes. RA is one of the participating factors in meiosis
and also in controlling differentiation into male or female phenotypes.
Low levels of RA generally prevent germline cells from entering
meiosis and increased levels induce meiotic resumption (Bowles
et al., 2006; Doyle et al., 2008). This is done through activation of
Stra8, Scp3 and Dmc1. While male gametes exposed to lower RA
concentrations stay quiescent until birth, female gametes exposed to
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Germ cells from stem cells
high RA concentrations enter the meiotic cycle with their arrival to the
genital ridge (McLaren, 2003).
The RA induces differentiation of ESC while stimulating the proliferation of precursors of the germ cells when added to the culture
medium. In vivo, the timing of germ cells entry into meiosis is dependent on the gonadal environment. Several lines of investigation suggest
that the ovary and testis have the same signalling system to induce
meiosis, but at different timing. RA signalling is the key regulator
responsible for the induction of germ cell meiosis in the fetal ovary
by inducing the expression of the germ cell-specific gene, stimulated
by RA, Stra8 (Bowles et al., 2006; Koubova et al., 2006). In contrast,
in the fetal testis this process is blocked by the metabolism of RA to an
inactive form mediated by the protein product of the Cyp26b1 gene
(Yashiro et al., 2004; Bowles et al., 2006; Koubova et al., 2006).
This gene is expressed in the early gonad of both male and female
embryos, preventing meiosis in the germ cells when they first arrive
at the gonad and come into contact with RA. When the somatic
sex is determined, the action of Sry leads to the up-regulation of
Cyp26b1 expression and the reduced activity of RA in the male
gonad, while it is down-regulated in the female gonad, where germ
cells respond to RA (Bowles et al., 2006). The source of RA in the
fetal urogenital region is the mesonephros (Bowles et al., 2006),
suggesting that the effect of RA inducing meiosis is direct. In the postnatal testis, it has been suggested that RA could be produced and
delivered by the Sertoli cells close to the spermatogonia, inducing
entry into meiosis in the same way as in the developing ovary, via
the up-regulation of Stra8 expression (Bowles and Koopman, 2007).
During murine and hESCs in vitro differentiation, markers of both
male and female germ cells have been detected in culture regardless
of the sex of the cell line (Hübner et al., 2003; Clark et al., 2004;
Lacham-Kaplan et al., 2006). The cells may be responding to an external signal instead to an intrinsic programme to enter meiosis. It is possible that RA present in the culture conditions propitiate meiosis
commencement and gamete determination. In fact, the bovine
serum added to cultures probably provides enough RA to induce
meiosis; media supplemented with 10% fetal bovine serum is estimated to contain 3.6 1028M RA (Fuchs and Green, 1981).
The most advanced progress in meiosis and formation of male
haploid gametes was obtained following transplantation of in vitroderived GSCs into the testis (Toyooka et al., 2003; Nayernia et al.,
2006b). Only two studies to date have explored co-culture systems
to establish oogenesis from ESCs in mice (Lacham-Kaplan et al.,
2006; Qing et al., 2007) and in both, established EBs were introduced
into biological systems. Lacham-Kaplan et al. (2006) explored the
effects of conditioned medium obtained from testicular cell cultures
of new born male mice on the appearance of germ cells within
mouse ESC-derived EBs. They discovered that a higher number of
EBs produce oocyte-like cells enclosed within follicular structures
when EBs were cultured in the conditioned medium. Appearance of
putative oocytes was also evident in EBs transferred to conditioned
medium at 120 h from initiation. These EBs already contained germ
cells, an indication that the formation of oocyte-like cells was dependent on the conditioned medium but not the appearance of germ
cells. Similarly, Qing et al. (2007) transferred established EBs onto
ovarian granulosa cell monolayer identifying oocyte-like cells within
the EBs after 10 days of culture. In both studies, the oocyte-like
cells did not contain the zona pellucida and appeared similar to
resting stage of in vivo gonocytes. Interestingly, the putative oocytes
obtained in both studies did not undergo spontaneous cleavage as
described by Hübner et al. (2003). The latter study described the
appearance of oocytes in differentiated mouse ESC cultures maintained in LIF-free medium with fetal calf serum.
Attempts to derive germ cells from human ES resulted in similar
findings as described in mice. Cells within hESCs-derived EBs
express markers for human germ cells including c-Kit, DAZL and
VASA; as well as SSEA-1 and Tra-1-60 (Clark et al., 2004; Aflatoonian
and Moore, 2005; Kee et al., 2006; Chen et al., 2007). This spontaneous differentiation is line-specific (Kim et al., 2007; Chen et al.,
2007). The addition of exogenous factors such as BMP-4, BMP-7
and BMP-8 to hESCs cultures increases the number of germ cells
expressing VASA, but does not necessarily induce their progress in
meiosis (Kee et al., 2006). From the few studies exploring the ability
of hESCs differentiation into germ cells, Chen et al. (2007) was the
only one to describe follicular-like structures appearing within monolayer or EBs of differentiated hESCs. Disappointingly, the study did not
explore the characteristics of cells enclosed within these follicular
structures to identify if they do indeed resemble oocytes.
It has been demonstrated that germ cells and gametes, both male and
female, can be differentiated from ESCs in culture. However, the efficiency of the different methods to obtain the precursors or early
germ cells is still low. The latest studies recently published develop
different strategies to increase the number of PGC-like cells to induce
the formation of mature gametes with higher efficiency. The different
protocols include strategies as prolonged cultures with low availability
of nutrients (Bucay et al., 2008), co-culture with mouse embryonic
fibroblasts in the presence of bFGF (West et al., 2008) and enrichment
of the population of PGC-like cells by FACS (fluorescence activated cell
sorting) using the marker SSEA1 (stage-specific embryonic antigen 1)
(Tilgner et al., 2008). In the last study, the authors reported that
obtained cells showed expression of markers such as Oct4, Stella,
VASA and SCP3, but no further differentiation was carried out.
Recently, a new study using a mESC line transfected with a reporter
gene associated with the GDF9 gene promoter (an oocyte-specific
gene) showed that GFP-positive cells with an oocyte phenotype
appeared surprisingly quickly in culture after differentiation in monolayer as well as through EBs formation (Salvador et al., 2008). The
authors suggest that a subpopulation of cells within the initial undifferentiated mESC might be already pre-programmed to undergo female
germline differentiation. These oocyte-like cells degenerated rapidly in
culture although evidence suggests that meiosis and parthenogenesis
may have occurred in some of them. Oocyte-specific structures
such as polar bodies and zona pellucida were observed, as well as
embryo-like structures with two or four cells. When LIF was added
to the culture medium the number of oocyte-like cells (GFP-positive
cells) significantly increased together with the expression of oocytespecific genes such as ZP3.
In vitro germ cells differentiation from
somatic SCs
Germline stem cells and mature gametes can be derived in vitro from
multipotent stem cells other than ESCs. In mice, teratocarcinoma and
mesenchymal stem cells (MSC) and induced iPS are considered multipotent cells, which are able to differentiate into various cell types
386
representative of all three germ layers (Kassem, 2004; Solter, 2006;
Takahashi and Yamanaka, 2006). With the exception of iPS cells,
the other cell types have been shown to differentiate into either germline stem cells or early germ cells in vitro and to advanced male
gametes in vivo following transplantation.
Embryonal carcinoma (EC) cells derived from teratocarcinomas, like
ESCs, are pluripotent and able to differentiate through EBs to germline
stem cells (Garcia-Sanz et al., 1996; Nayernia et al., 2004). When
transplanted into sterile mice, EC-derived germline stem cells are
able to complete meiosis and spermatogenesis (Nayernia et al.,
2004). Regardless of these promising abilities, sperm numbers in the
ejaculate are reduced and 43% are morphologically abnormal (compared with only 8% in normal males). Sperm are able to fertilize
oocytes through intracytoplasmic injection but the resultant zygotes
proceed to the 6 –8-cell stage only.
Teratocarcinoma-derived EC cells have similar imprinting patterns as
PGCs. As such, the chromatin is globally hypomethylated and the promoter of imprinted genes such as H19 is unmethylated (Okamoto and
Kawakami, 2007). Hence, global methylation erasure is not required
when teratocarcinoma cells are used to derive GSCs in vitro.
It appears, therefore, that re-establishment of imprinting in postmeiotic gametes obtained from teratocarcinoma-derived EC cells is
also unsuccessful, resulting in preimplantation embryo developmental
abnormalities.
MSCs obtained either from the bone marrow (BM) or alternatively
from fat tissue, were also shown to differentiate into several lineages
(Kassem, 2004). It has also been proposed that MSCs are progenitors
for oocytes in adult ovarian tissue (Johnson et al., 2005). That latter
revolutionary proposal has been regarded as unreliable and has
sparked controversy and several solid arguments have been raised
against it (Byskov et al., 2005; Eggan et al., 2006). A recent work published by Liu et al. (2007) showed that meiosis, neo-oogenesis and
GSCs are unlikely to occur in normal adult human ovaries. If post-natal
oogenesis is finally confirmed in mice, then this species would represent an exception to the rule. Stronger evidence is needed to
confirm this new theory indicating that these somatic SC-derived
oocytes enter meiosis or support the development of offspring in
cases of patients with allogenic BM transplant.
However, the authors of these controversial studies have come into
discussion refuting the arguments against post-natal oogenesis in adult
human ovary arguing, among others, that this reasoning derives from
the inability of the authors to detect markers of germ cell mitosis
and meiosis, and that an absence of evidence does not mean an
absence of the possibility (Tilly and Johnson, 2007). Curiously, a
recent work published by the same group presents a mouse model
in which BM transplant helps to preserve or recover ovarian function
of recipient females, but all offspring generated derived from the host
germline and not from the transplanted BM cells (Lee et al., 2007).
Nayernia et al. (2006a) demonstrated that mouse MSCs are able to
give rise to germline stem cells in vitro. As for germline stem cells derived
from teratocarcinoma and ESCs, MSCs-derived germline stem cells
arrest at premeiotic stages upon transplantation into the testes of
adult sterile mice. More recently, this group announced
the differentiation of sperm from human BM-derived stem cells
(Drusenheimer et al., 2007). However, they have no ability to
examine the functional competence of these cells, the successful establishment of which will remain unexplored due to ethical constrains.
Marques-Mari et al.
Attempts to develop potential germ cells from somatic cells were
demonstrated by Dyce et al. (2006) who have been able to differentiate oocyte-like cells from fetal porcine skin. Skin SCs in this study
were isolated and cultured in follicular fluid with the addition of
exogenous gonadotrophin. This resulted in the formation of follicular
structures containing putative oocytes. The oocyte-like cells underwent spontaneous cleavage in culture similar to that described by
Hübner et al. (2003). While it remains unclear if the skin SCs
de-differentiated into ES-like cells before differentiation into the germline the authors postulated precociously that these events are similar
to those described by Johnson et al. (2005) in mice. In humans,
Bukovsky et al. (2005) reported that new oocytes can be derived
from ovarian cortical mesenchymal cells. While as previously discussed, mice BM MSCs can give rise to germ cells in vitro, it has not
been proven that human MSCs from the BM or any specific tissue
are able to do so. On the contrary, Liu et al. (2007) clearly demonstrated that early meiotic-specific or oogenesis-associated mRNAs
for SPO11 (involved in the early steps of meiotic prophase, Shannon
et al., 1999), PRDM9 (essential for progression through early
meiotic prophase, Hayashi et al., 2005), SCP1 (necessary for establishment of synaptonemal complex, Yuan et al., 2000), TERT (role in stem
cells proliferation, Martin-Rivera et al., 1998) and NOBOX (required
for follicle development, Rajkovic et al., 2004) were undetectable in
adult human ovaries using RT –PCR, compared with fetal ovary and
adult testis controls. These findings are further corroborated by the
absence of early meiocytes and proliferating germ cells in adult
human ovarian cortex probed with markers for meiosis (SCP3),
oogonia (Oct4, c-Kit) and cell cycle progression (Ki-67, PCNA), in
contrast to fetal ovary controls.
In summary, when male gametes differentiation has been
attempted, the best results have been obtained with transplantation
of premeiotic germ cells selected from differentiated EBs into in vivo
systems (Toyooka et al., 2003; Nayernia et al., 2006b). For female
germ cells differentiation, both culture in monolayer and through
EBs formation succeeded although the oocyte-like cells obtained
seemed to be in an immature stage, in most cases with a fragile structure. Their use for the generation of live progeny has not been
reported.
The addition of exogenous factors to the culture media such as
BMPs (Toyooka et al., 2003; Kee et al., 2006) or RA (Geijsen et al.,
2004; Nayernia et al., 2006b), which play basic roles in germ cells
and gametes development in vivo, seem to help to expand the germ
cell population and push them to the meiotic process in vitro, but it
is not enough to direct them through that process properly. At this
point, a gonadal-like three-dimensional structure or specific cell-to-cell
contact might be required to progress through meiosis and to acquire
a correct epigenetic status. An environment similar to the in vivo niche
might be a necessary requisite.
Demanding biological descent:
how to obtain patient-specific
gametes
The growing demand for biological offspring for patients with impaired
fertility has put its hope in scientific research and the obtaining of
patient-specific differentiated gametes. Theoretically, it can be
387
Germ cells from stem cells
accomplished through two different techniques: somatic cell nuclear
transfer (SCNT) and reprogramming. In both cases, the newly
derived gametes will be genetically identical to the individual whose
gametes are trying to be replaced.
Nuclear transfer can be defined as the creation of somatic embryos
(no gametes are involved) using the oocyte cytoplasm as the reprogramming conductor following transfer of somatic nuclei into enucleated mature oocytes. In this case, hESCs can be derived from
these somatic embryos and differentiated gametes will match the
adult individual.
Compared with the hundreds of published reports on animal
SCNT, few studies to date have been published on human SCNT
(Cibelli et al., 2001; Stojkovic et al., 2005; Lavoir et al., 2005).
Results from these studies indicate that human SCNT is a highly inefficient technique that often results in poor embryonic development
with high aneuploidy rates. No hESCs obtained from SCNT human
embryos have been reported so far, although very recently successful
derivation of primate ESCs from cloned embryos has been reported
by Byrne et al. (2007), and the genotyping of these Rhesus SCNT pluripotent cell lines was verified by an independent group (Cram et al.,
2007). These new reports encourage the continuation of the use of
SCNT for therapeutic purposes.
To overcome ethical, social and legal problems, iPS cells may, in the
future, replace ESCs for clinical therapies. iPS cells are fibroblast cells
from mouse or human tissue that are reprogrammed to the pluripotent state by introducing factors into their genome known to induce
pluripotency such as Oct4, Sox2, c-Myc and Kif4 through retroviral
transfection (Takahashi and Yamanaka, 2006; Maherali et al., 2007;
Takahashi et al., 2007; Wernig et al., 2007). This simple and revolutionary approach might overcome the use of oocytes and the production of somatic embryos as a source for ESC lines. This new
type of stem cell, iPS, resemble ESCs by morphology and growth
properties, expression of ESCs marker genes and teratoma formation
(Takahashi and Yamanaka 2006; Maherali et al., 2007; Takahashi et al.,
2007; Wernig et al., 2007). However, their global gene expression and
DNA methylation patterns are similar but not identical to that of
ESCs. Three studies presented a second generation of iPS adding a
new factor (Nanog) to the cells (Maherali et al., 2007; Okita et al.,
2007; Wernig et al., 2007). The selection of Nanog resulted in germline competent iPS cells leading to the formation of chimeric mice.
A high proportion (20%) of chimeric mice developed tumours resulting from the c-Myc gene expression (Okita et al., 2007), which eliminates the possibility of their current use as prospective germline stem
cell progenitors.
Applications and future
prospects
Seemingly, the germ cell differentiation process is dependent on spatial
distribution of differentiating cells. EBs represent a three-dimensional
structure with a microenvironment that propitiates differentiation,
although other cell types besides germ cells arise from them
(Itskovitz-Eldor et al., 2000).
Currently, one of the most critical steps after differentiation is selection and isolation of differentiated germ cells. Since ESCs and PGCs
share some common markers, detection of post-migratory and
meiotic markers is a useful method, as well as the use of transgenes
with fluorescent reporter genes under the control of specific promoters of male and female germ cells. These have been the most
employed strategies in the studies of germ cell differentiation developed to date (Hübner et al., 2003; Toyooka et al., 2003; Nayernia
et al., 2006b). The problem is that methods based on gene modification and the use of retroviral vectors limit the use of gamete-like
cells in future clinical treatments.
Future translational application of these ESC-derived gametes in
ART when functional gametes are not available from patients
require still further investigation into gamete differentiation. Ultimately,
the process needs to be reproducible and efficient. The focus of
current and future studies should be on meiotic completion to avoid
unwanted aneuploidies and the determination and establishment of
accurate epigenetic modifications and imprinting status to provide
reproductive hope to humans lacking gametes.
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Submitted on November 15, 2007; resubmitted on December 5, 2008; accepted on
December 31, 2008