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EMBRYONIC STEM CELLS-CHARACTERIZATION SERIES: CONCISE REVIEW |
a Early Human Development and Stem Cells Group,
b Human Genetics Division,
c Developmental Origins of Health and Disease Division, University of Southampton, Southampton General Hospital, Southampton, United Kingdom
Key Words. Human • Embryonic germ cell • Primordial germ cell • Stem cell • Embryo • Gonad
Correspondence: Neil A. Hanley, M.R.C.P., Ph.D., and Lee Turnpenny, Ph.D., Human Genetics Division, University of Southampton, Duthie Building (M.P. 808), Southampton General Hospital, Tremona Road, Southampton SO16 6YD, United Kingdom. Telephone: 44-0-23-8079-6421; Fax: 44-0-23-8079-4264; e-mail: N.A.Hanley{at}soton.ac.uk
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ABSTRACT |
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INTRODUCTION |
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). The inner cell mass (ICM) of the early embryo gives rise to the derivatives of all three germ layers in the developing embryo. Taking the ICM into in vitro culture offers the opportunity to derive embryonic stem cells (ESCs). These cells, first attained from mouse embryos by Evans and Kaufman [1] and, independently, by Martin in 1981 [2], retain the ability for broad differentiation but also undergo self-renewal. As such, they are the pluripotent stem cell that has been the focus of most research. An alternative source of pluripotent cells arises later in development. Germ cells are the sole means of transmitting genetic information to the next generation in their ultimate form as haploid gametes, spermatozoa, and ova. However, before meiosis, these cells exist as diploid primordial germ cells (PGCs). PGCs share significant similarities to the cells of the ICM and, once taken into in vitro culture, can lead to the generation of embryonic germ cells (EGCs) (Fig. 1), the parallel of ESCs. The derivation of human EGCs (hEGCs) has been reported now by several groups worldwide [3–6]. This review brings together these experiences and compares their emerging biology with that of human ESCs (hESCs), beginning from the historical starting point of PGC-EGC studies in mice.
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Understanding the biology and proliferation of mammalian PGCs requires appreciation of a fascinating journey from their origin in the epiblast, adjacent to the extraembryonic ectoderm, through the gut mesentery, to their final destination in the developing gonad (Fig. 2) [13]. Although the mechanisms regulating early mobilization are unclear, or even random, later migration relies in part on the chemokine Sdf-1 released from somatic cells. In mice, these signals act on the PGC surface receptor Cxcr4b to activate phosphatidylinositol 3 kinase and G-protein–coupled pathways that are responsible for PGC motility and direction, respectively [14–16]. In addition to chemoattraction, migrating germ cells require other stimuli for their maintenance, such as stem cell factor (SCF) (also known as steel factor, mast cell growth factor, or Kit ligand), acting via its receptor c-Kit. Disruption of this signaling pathway leads to germ cell apoptosis and a failure of PGCs to reach the gonadal ridge [17].
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Knowledge of human PGCs (hPGCs) is more difficult because they are less amenable to study. Germ cells are apparent in the gonadal ridge during the fifth and sixth week of development, with further PGCs detected in the gut mesentery, most likely in transit (Fig. 2
). By 41 to 44 dpc (Carnegie stages 17 and 18), Sertoli cell differentiation and testicular cord formation is associated with decreased numbers of PGCs in male compared with female embryos, presumably due to mitotic arrest [22]. In contrast, proliferation continues in the developing fetal ovary during the remainder of the first trimester. This ability for continued mitosis in female fetuses carries potential significance. Female menopause results from an exhausted supply of gametes and is considered premature if prior to 40 years. One hypothesis to explain this untimely ovarian demise is inadequate provision of germ cell number before meiosis. It becomes plausible, therefore, that genes associated with premature ovarian failure are candidate regulators of PGC proliferation and vice versa.
Mouse EGC Derivation
The identification of mPGCs fostered attempts to isolate and study their properties in vitro. Survival depended on age: PGCs from 13.5 dpc onwards could be cultured for days, with female cells entering meiosis. In contrast, cells at 11.5 to 12.5 dpc did not survive unless temperature was reduced to 30°C, favoring continued mitosis rather than meiosis of female germ cells [23]. Even so, cells survived little more than 1 week, either succumbing to apoptosis or differentiating. Problematic to these cultures was the apparent aversion of germ cells to standard cell culture substrates (e.g., plastic, glass, and gelatin). In contrast, gonad explant cultures demonstrated greater survival of PGCs when supported by outgrowing, adherent somatic cells. Hence, growth-arrested, embryo-derived feeder cell monolayers were adopted as an effective adherent substratum for PGCs [24]. This more reliable culture support system allowed distinction between pregonadal migratory and gonadal postmigratory PGCs. Culture behavior mirrored events in vivo: later stages yielded fewer motile PGCs, especially in male cells [25]. However, it was evident that some migratory cells retained mitotic activity in culture, albeit for a limited period.
Survival and proliferation could be promoted by the synergistic action of SCF and leukemia inhibitory factor (LIF), although proliferation did not progress beyond that of their in vivo counterparts [26–28]. Critically, extra supplementation with fibroblast growth factor 2 (Fgf2) effected their continued proliferation beyond normal in vivo cessation, a criterion in defining conversion or derivation from PGCs to EGCs [29, 30]. Remarkably, these more persistent cells possessed properties redolent of the mouse ESC (mESC) lines first established 10 years earlier, satisfying all criteria for pluripotency, both in vitro (giving rise to embryoid bodies [EBs] containing derivatives of all three germ layers) and in vivo (contribution to all tissues and the germ line of chimeric animals). Subsequently, derivation has been proven most successful from PGCs before and during migration but remains attainable from PGCs within the gonadal ridge until 12.5 dpc [31]. After this time, presumably mitotic arrest in male PGCs and the first stage of meiosis in female cells renders them refractory to EGC derivation. This contrasts to a population of multipotent germline stem (MGS) cells that has been isolated recently from the mouse neonatal testis, most probably derived from the diploid spermatogonial stem cell population [32].
Human EGC Derivation
The knowledge acquired over the years from mPGC-mEGC culture was a sensible starting point for attempts to derive hEGCs. However, in contrast to the plethora of laboratories that have derived hESC lines, reports of hEGC derivation remain limited. The first report (soon after the initial description of hESCs in 1998 [3, 33]) has been reinforced by three additional groups (ourselves in 2003 and two other groups in 2004, the latest group reporting twice with slightly varied methodology [4–6, 34]). This restriction may be due in part to the complications in acquiring ethically approved, continued access to the required starting material but also doubtless reflects the difficulties in the derivation and culture management of hEGCs, as acknowledged by some groups [4, 5, 35]. Nevertheless, this progress demonstrates that the process is practically, rather than just theoretically, possible.
The Starting Human PGC Population
The nature of acquiring human material from first-trimester voluntary/social termination has restricted the age of the PGCs available. This material spans 5 to 9 weeks after conception, with no bias evident between groups from the use of the anti-progestogen mifepristone/RU486 versus surgical termination of pregnancy (Table 1). The earliest specimens would contain PGCs shortly after arrival in the gonad, which might be expected to maintain proliferation better in culture; however, there has been little evidence to support this. Whereas there is an apparent upper age limit for mEGC derivation of 12.5 dpc in both males and females, it is remarkable that an upper limit related to sex cord formation cannot be applied to human male PGCs, as all reports include derivation from the fetal testis after this event. There are several potential explanations for this: male PGCs might resume proliferative activity once freed from Sertoli cell influence; cord formation may not arrest all male PGCs (i.e., a small cohort of proliferative PGCs might persist); or use of the entire urogenital ridge, as reported by most groups [3, 5, 6, 34], might include a population of extragonadal PGCs. Our continuing investigation considers both the retention of mesonephros, which contains some alkaline phosphatase (AP)-positive cells [4], and its removal from the fetal gonad. To date, no significant difference is apparent.
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). We have compared enzymatic dissociation with puncturing the intact organ, as developed initially for the isolation of mPGCs [36]. Equally effective at generating hEGC cultures, cells are released directly into culture media, avoiding protracted washing and resuspension. At least theoretically, this avoidance of proteolytic enzymes minimizes damage to cell-surface markers and receptors during initial preparation and plating.
Basic In Vitro Culture Media
In reports to date, the basic composition of culture media has shared many similarities to mEGC derivation methodology, comprising a mix of Dulbecco’s modified Eagle’s medium (DMEM) or knockout DMEM (KO-DMEM) with nonessential amino acids, beta-mercaptoethanol, and L-glutamine. Choice of serum varied between 10%–15% fetal bovine serum (FBS), ESC-tested FBS, or knockout serum replacement (KO-SR) (Table 1).
Whereas serum provides essential nutrients, its use is far from ideal, being the primary source of unknown factors with the potential to affect derivation or induce differentiation. Alternatively, the serum-free supplement KO-SR can (in conjunction with KO-DMEM) decrease the propensity for spontaneous differentiation of mESCs [37] and can improve the derivation efficiency of mEGC, although growth is reduced [38]. Its effects on the efficiency of hEGC derivation remain conjecture due to the relatively unpredictable nature of these cells in culture, with or without serum.
Feeder Layers
The reporting groups have predominantly used either mouse STO fibroblasts or primary mouse embryonic fibroblasts (pMEFs) as feeder layers (Table 1). Shamblott et al. [3] are the only researchers to date who have reported the comparison of feeder layers. Although in their experience STO cells were preferable to either pMEFs, human fetal fibroblasts, or gelatin-coated plates, further formal analyses of feeder layer characteristics, such as via proteomic approaches [39], are required to discern between the factor cohorts. This is particularly important given the complexity of in vivo PGC-somatic cell interaction within the fetal gonad and the consequences of alien interactions, such as the in vitro meiotic influence of the fetal lung on mPGCs [40]. Similar to this experience, it is conceivable that different cell types, providing differing factor combinations, will improve the sequential derivation and maintenance of hEGCs.
Media Additives and Critical Factors
In addition to the influence of the basic media and feeder layers, all groups reporting hEGC derivation and culture have included other additives. Their use originates from mouse pluripotent stem cell derivation and culture. Definitive requirements for any in equivalent hEGC cultures have yet to be established conclusively.
Stem Cell Factor. Perhaps the role of SCF is the most understandable. The sterile mouse mutants Sl (Steel) and W (Dominant White Spotting) arose from mutations affecting SCF and its receptor, c-Kit, respectively, and revealed a direct role in the proliferation and maintenance of PGCs en route to the gonad [41]. c-Kit is present on the cell surface of PGCs. Its ligand, SCF, exists as two isoforms: a membrane-bound (mbSCF) factor and a soluble form (sSCF), produced by proteolytic cleavage. mbSCF is critical for the proliferation of PGCs in vivo [42]. In vitro, it promotes mPGC survival by inhibition of apoptosis for longer than sSCF [17, 26, 43, 44]. The latter isoform, however, increases telomerase activity in vitro [45]. It is therefore easy to envisage the benefit of both sSCF supplementation combined with a feeder layer, such as STO, supplying mbSCF [46]. As in the mouse, c-KIT and SCF are also present in human fetal gonads [47] (our unpublished data).
Leukemia Inhibitory Factor. The role of LIF is intriguing. PGC development in LIF-deficient mice is normal, suggesting either no role or redundancy between related cytokines. Indeed, oncostatin M can substitute for LIF in affecting survival and/or proliferation of mPGCs in culture [46, 48, 49], although neither can substitute for feeder support [46]. In contrast, LIF, originally isolated from buffalo rat liver (BRL) cells for its differentiation inhibiting activity [50], can obviate the requirement for feeder layers in mESC culture, where signaling via the LIF receptor (LIFR), gp130, and intracellular Stat3b has a proven role in maintaining mESC pluripotency [51].
There are differences in pluripotent stem cells between mice and humans. Despite activation of the LIFR/gp130-STAT3B pathway, LIF (administered in human recombinant form) does not maintain self-renewal of hESCs, which require feeder cells or their conditioned media with an extracellular matrix [52, 53]. However, this highly pleiotropic cytokine may have earlier effects. In LIF-deficient mice, blastocysts fail to implant [54]. The receptors LIFR and gp130, although dispensable for normal early embryogenesis, are necessary for diapause, the suspension of mouse development at the blastocyst stage in the event of unfavorable conditions. Under these circumstances, the ICM can maintain its undifferentiated state for months [55]. Although human embryos do not enter such a phase, LIF increases the number and quality of human blastocysts in serum-free culture [56], and medium conditioned by BRL cells potentially improves hESC derivation [57].
In PGCs, however, the effects of LIF are debatable. LIFR/ gp130/Stat3b signaling inhibits the progression to meiosis of cultured female mPGCs [58], but this has not been reported in human cells. Similarly, although all groups have included LIF during hEGC derivation and culture, none have systematically proven its necessity. Certainly LIF, LIFR, gp130, and associated signaling components are expressed in human fetal gonads [59] (our unpublished data). Perhaps active LIFR/gp130/Stat3b signaling in vivo sustains hPGCs in an undifferentiated suspended state (akin to diapause). If this is the case, once removed and exposed to myriad other, as-yet-undefined influences in vitro, this effect may be overridden, resulting in the loss of hPGCs by cell death and/or differentiation (including meiosis).
Fibroblast Growth Factor 2. As described earlier, Fgf2 was the key addition that enabled mPGCs to continue proliferation in cultures already containing LIF and SCF [60]. It is unclear specifically which Fgf receptors transduce its effects, but the receptivity of mPGCs is important. Potentially, only transient (approximately 12-hour) exposure to Fgf2 is necessary for mEGC derivation [41], after which its supplementation, along with SCF, can be discontinued so long as cells are maintained on feeders in the presence of serum [29]. However, after 11.5 dpc, reduced Fgf receptor expression also correlates to diminished derivation efficiency. This effect is more pronounced in females, perhaps due to the approach of meiosis [60, 61].
Fgf2 functions as a potent mitogen in many cell types and induces telomerase activity in cultured mouse neural precursor cells [62]. This suggests overlap or synergism with SCF in maintaining telomerase activity of mPGCs when taken into culture.
Forskolin. All groups deriving hEGCs have included forskolin, which raises intracellular cAMP levels and stimulates mitosis in cultured mPGCs [63]. Whether this action compensates for an endogenous hormone is not known; however, in mEGCs it does not substitute for the survival mediated by LIF and SCF [64].
Although included by the groups reporting hEGC derivation, systematic definition of optimal concentrations and time exposures of all these and other growth factors remains a significant challenge for hEGC researchers. Furthermore, the recent description of mouse MGS cells arose from cultures that primarily maintained spermatogonial stem cells, i.e., the self-renewing cells that give rise to spermatozoa [32]. These cultures included glial-derived neurotrophic factor (GDNF) and epidermal growth factor (EGF). Whereas MGS cells did not develop in standard mEGC derivation media (LIF, SCF, and Fgf2) [32], the converse effect of GDNF and EGF on mouse or human EGC derivation and maintenance is unknown.
Growth Characteristics of Human PGCs and EGCs
The essence of hPGC-EGC basic biology can be summarized as overcoming the in vitro sensitivity of PGCs that have been removed from their specialized in vivo niche, the continuation (or reacquisition) of proliferation, and the maintenance of self-renewal and pluripotent properties in prolonged culture.
As with mouse, culture as gonadal explants mimics the supportive in vivo environment for hPGCs, but outgrowth is limited (Fig. 3). Dissociation and replating isolates the germ cells, but the consequences are not all beneficial. The fetal gonad is heterogeneous. In vivo, germ cells develop supportive intercellular contacts with specialized somatic cells, such as Sertoli (nurse) or granulosa cells. The loss of supportive in vivo paracrine factors is detrimental, yet other somatic influences (e.g., from the Sertoli cell) inhibit proliferation [20]. In stark contrast, culturing the ICM benefits from maintained intercellular contact with the same cell type.
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). Neither resembles colonies of hESCs. Although PP cultures have survived with AP+ cells beyond 50 days, these cultures have never converted to intense proliferation, despite factors such as Fgf2. The 15% of cultures that rapidly proliferate tend to do so early—typically within the first 2 weeks in culture. This suggests that the problem of survival is less important than that of conversion to a cell with VP/hEGC characteristics. Information on the molecular differences between preconversion and postconversion germ cells within the same and across different species would be instructive.
Several groups, including ourselves, have noted difficulty in maintaining hEGCs undifferentiated long-term [4, 5, 35]. This problem of undifferentiated status contrasts with other pluripotent stem cell types: hESCs and human embryonal carcinoma cells (hECCs) and mESCs and mEGCs, all of which have been more extensively characterized. In our experience, VP hEGC cultures have proliferated extensively; however, the proportion of cells expressing pluripotent markers (e.g., OCT4 and stage-specific embryonic antigen [SSEA] family members; see below) declines over time, variably from 2 to 3 months onwards, and is exacerbated by freeze-thaw routines [4]. In striking contrast, a single report from Park et al. [6] described continuous, undifferentiated culture of hEGCs for 12 months, despite similar methodology and starting material (Table 1). All human pluripotent cell types are continuously prone to spontaneous differentiation, and their properties, including karyotype, can alter with high passage number [66]. This group’s experience needs to be shared by other researchers, including characterization of whether the properties of this line can be retained through freeze-thaw cycles.
In Vitro and In Vivo Characterization of hEGCs
hEGCs have been subject to the same tests of stem cell status as hESCs and hECCs. In addition, loss of pluripotent markers merits greater consideration of meiotic progression as well as normal somatic differentiation [58].
Gene Expression Analyses
The self-renewal of karyotypically normal hEGCs has been assessed by the expression of characteristic markers (Table 2 and references therein). All groups report AP activity. SSEA1 and SSEA4 are present; however, groups either report variable SSEA3 expression or its absence. It remains unclear what significance should be attached to the expression of SSEA family members. In hESCs and hECCs, SSEA1 is absent until the onset of differentiation, whereas SSEA3 is readily detected. In contrast, hEGCs start out SSEA1-positive, with, at best, only weak SSEA3 immunoreactivity (Table 2). Although this profile might suggest early differentiation, the strongly SSEA1+/EMA-1+ starting population of PGCs within the gonad argues against this [4]. It is also difficult to be certain of the significance of these differences when the genes under discussion perhaps mark, but do not regulate, cell phenotype. Conversely, genes and proteins with known function in pluripotent cells serve as more informative markers. Particular salient examples are the nuclear transcription factors OCT4 and NANOG, which have key roles in the maintenance of pluripotency [67–69] and are expressed in hPGCs [4, 70]. All groups have identified OCT4 in hEGC by reverse transcription–polymerase chain reaction and/or immunocytochemistry. Some groups have identified telomerase activity either by telomerase repeat-amplification protocol assay or the expression of hTERT. Additional genes, such as SOX2, FGF4, STELLAR, FRAGILIS, or DDX4, highlighted in ESCs or as orthologs in the germ cell lineage of other species, have not been characterized in either hPGCs or hEGCs. The expression of STELLAR is relevant as it forms part of the cluster at 12p13 that includes NANOG and GDF3, which is frequently overrep-resented in teratocarcinogenesis and contained within 12p iso-chromosomes in several aneuploid EC lines [71, 72].
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). The significance of hTERT, present within hPGCs and hEGCs, is less clear. This catalytic component of the telomerase ribo-nucleoprotein is a marker of nonsenescing cells, which maintain telomere length. This includes stem cells, but also other cell types, such as cancer cell lines or those from human fetal development (i.e., postgastrulation) [73]. Nevertheless, loss of this gene expression profile coincides with the onset of differentiation via haphazard EB formation to ectodermal, endodermal, and mesodermal end points. This satisfies in vitro criteria for pluripotency and has been achieved by all four groups who have derived hEGCs [3–6]. Unlike ESC-derived structures, however, no spontaneous contractions consistent with mesodermal differentiation toward cardiomyocytes have been reported. Furthermore, since the original report, which maintained the presence of LIF, Fgf2, and forskolin [3], the subsequent groups have all withdrawn LIF and, in some instances, the other factors from the culture medium [4–6]. These apparently conflicting observations, possibly due to factors intrinsic or extrinsic to hEGCs, or a combination of both, require clarification. The issue of hEGC pluripotency becomes particularly interesting with in vivo analyses, for which the only true test is the ability to form chimeric embryos, not permitted with human cells for ethical reasons. By these criteria, mouse ICM cells, mESCs, mEGCs of either sex, and the recently derived mouse multipotent germline cells (mMGCs) can give rise to all somatic cell lineages, as well as functional gametes [32, 74–76]. In contrast, mPGCs are nullipotent [41]. Without recourse to these experiments for human cell types, teratoma formation is taken as standard evidence of pluripotency in vivo and has been achieved for hESC and hECC [41]. Further, this property (unlike efficiency of derivation) is not dependent on genetic background [74, 75]. Intriguingly, no group has reported teratomas from hEGCs. Our experience has been to engraft early-passage hEGCs into the thigh muscle of immunocompromised mice. The cells expressed AP at the time of engraftment. Whereas the positive control of human N-TERA2 ECCs, cultured in parallel, yielded characteristic teratomas, no such tumors have been observed from equivalent numbers of hEGCs, consistent with findings elsewhere [77]. The multiple cell lineages within a teratoma confirm pluripotency. However, failure to form a tumor does not mean hEGCs lack pluripotency. Teratomas arise from unrestricted local proliferation of the undifferentiated cells that then differentiate to derivatives of all three germ layers. Our preliminary evidence suggests that at least some human cells persist within the immunocompromised mice, but their phenotype is, as yet, unclear. This insinuates that hEGC proliferation was restrained sufficiently to avoid tumorigenesis, but potentially in its place cells have differentiated (if so, presumably in response to cues from their respective individual murine environments).
So, at present, in the absence of the definitive chimeric experiment, the in vivo pluripotency of hEGC remains unresolved. Two therapeutic considerations may lessen the importance of this: first, in vivo transplantation of EGC-derived cells has generated functional responses; second, lack of teratoma formation is highly desirable for therapy in human recipients.
Therapeutic Potential of Human EGCs
Important studies toward therapeutic exploitation of hEGCs have progressed. The in vitro differentiation capacity of hEGC, via ongoing culture of EB-differentiated (EBD) cells, has been well described by the original deriving group [78], including in vitro characterization in response to BMP2 and transforming growth factor ß3 of a musculoskeletal phenotype [79]. EBD cells, capable of significant expansion in culture, also provided the material to demonstrate that imprinting appears appropriate with monoallelic expression of several relevant genes, a critical consideration for potential therapeutic applications [35]. Further accumulating evidence reflects a propensity for neuronal differentiation [6, 34] (our unpublished data). Pan et al. [34] found that, as for hESCs, retinoic acid or increases in intracellular cAMP signaling enhanced neural differentiation of hEGC derivatives, including tyrosine hydroxylase-positive cell types relevant to therapy for Parkinson’s disease. Whereas this study was restricted to in vitro investigation, cellular derivatives of hEGCs have also improved motor function in rats in vivo. Interestingly, after diffuse motor neurone injury, the grafts of transplanted human cells did not directly reinnervate tissues; instead, they appeared to support the survival and regrowth of endogenous rat neurones [80]. Taken together, these studies argue for the inclusion of hEGCs in stem cell research programs.
Future Directions
Now that several groups have successfully established hEGC cultures, an increase in protocols for directed differentiation is likely to follow. The goals of these experiments are no different from other aspects of human stem cell research: namely, to generate physiologically normal cells with the desired function, ideally by normal regenerative or developmental pathways. It remains to be seen whether the undifferentiated cells ever produce teratomas. Potentially, this will be an unforeseen advantage of hEGC research, in which case-comparative study of hEGCs and hECCs also offers an informative model on the origin of gonadal tumors.
Better understanding is needed of the derivation process and long-term culture so that true robust hEGC lines can be banked equivalently to hESCs. This needs to include transition to human feeder cells, or, ideally, their complete avoidance, and gearing up of cell culture efforts toward good manufacturing practice. To achieve these goals, our current focus is on trying to understand the determinants of derivation. For this, better knowledge of the starting hPGC population will allow definition of the differences that arise between species and after derivation. Unique to PGC-EGC research, this requires consideration of the complex somatic-germ cell interactions that differ between male and female and the propensity for meiotic differentiation. All of this research will be hindered without refined methods for isolating pure PGC populations away from their somatic neighbors. Conversely, achieving this will allow better gene expression analyses and standardized culture experiments that enable more thorough analysis of additive factors and comparison of EGCs with ESCs and ECCs. It will be revealing to determine whether the differences apparent at the cell surface, currently demonstrated by the variable requirement for exogenous factors, translate to intracellular differences or whether the same underlying molecular pathways are truly common to all human pluripotent stem cells.
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ACKNOWLEDGMENTS |
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DISCLOSURES
The authors indicate no potential conflicts of interest.
NOTE ADDED IN PROOF
Since electronic publication of this article, a further group has reported on aspects of the human PGC-EGC lineage [82] and their use in cell therapy.
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