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作者：Lee Turnpennya,b, Cosma M. Spallutoa,b, Rebecca M. Perretta,b, Marie OSheaa,b, Karen Piper Hanleya,b, Iain T. Camerona,c, David I. Wilsona,b, Neil A. Hanleya,b
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# _( {: _1 [* o |, p. ~3 l0 v0 K 【摘要】6 J5 e1 d# F( ~* v9 J/ u
The realization of cell replacement therapy derived from human pluripotent stem cells requires full knowledge of the starting cell types as well as their differentiated progeny. Alongside embryonic stem cells, embryonic germ cells (EGCs) are an alternative source of pluripotent stem cell. Since 1998, four groups have described the derivation of human EGCs. This review analyzes the progress on derivation, culture, and differentiation, drawing comparison with other pluripotent stem cell populations. : Q2 d) M% A7 i4 _& `; K
【关键词】 Human Embryonic germ cell Primordial germ cell Stem cell Embryo Gonad+ ^( P" N! T& h. o! n
INTRODUCTION
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In mammalian biology, two clear sources of untransformed pluripotent stem cell have been described (Fig. 1). 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 . 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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Figure 1. Cartoon of human embryonic stem cell and embryonic germ cell derivation. Abbreviations: hEGC, human embryonic germ cell; hESC, human embryonic stem cell; ICM, inner cell mass; PGC, primordial germ cell.
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, Q. l- X% \+ m: _" |# l& SOrigin, Migration, and Proliferation of PGCs9 x# g6 P: t8 I$ l1 _2 f: J0 ]" w
/ d2 z9 a5 w7 |; z0 p6 gTo understand EGCs, it is first necessary to comprehend how PGCs arise and proliferate and distinguished from the surrounding somatic cells by a longer cell cycle (16 versus 7 hours).; h/ x& n7 ], Q: Z' H i
; e: b1 v! n; x5 [; qUnderstanding 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) .
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* ]2 X. g% d, @! |/ z xFigure 2. Migration of human primordial germ cells. Representation of human primordial germ cell (PGC) migration from the allantois to the gonadal ridge in the intact embryo (A) and through the gut mesentery within the dissected abdomen (B) at approximately 6 weeks after conception. The gonadal ridge (G) has developed on the medial surface of the mesonephros (M) adjacent to the adrenal gland (A) and superior to the kidney (K). (C): Human embryo section corresponding to (B) showing PGCs darkly stained for alkaline phosphatase activity in the gonad (G) and throughout the folds of the gut mesentery (arrow). Bar = 250 µm.0 R0 s5 M4 a# |, O8 j" U7 y9 @+ D+ X
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Between 10.5 and 12.5 days postconception (dpc) in mice, PGCs arrive at the gonadal ridge, and those that fail regress . In both sexes, arrival in the gonadal ridge also heralds the final erasure of methylation patterns.1 O' E& ^2 c% ?5 C4 W
% P4 D, V& q/ U) S8 h nKnowledge 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 . 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.
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Mouse EGC Derivation6 ?6 W+ u% N0 _- E' {3 p n
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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¡ãC, favoring continued mitosis rather than meiosis of female germ cells . However, it was evident that some migratory cells retained mitotic activity in culture, albeit for a limited period.' C7 t" ]( a* G
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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 ., s7 l' S. H$ `; |
* X& f s/ h9 v4 S0 gHuman EGC Derivation9 o& G( K3 J, O( I0 o
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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 . Nevertheless, this progress demonstrates that the process is practically, rather than just theoretically, possible.! ]' N! G+ o: H
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The Starting Human PGC Population
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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 , and its removal from the fetal gonad. To date, no significant difference is apparent.
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, x) Y* C4 C2 ] CTable 1. Methodology in published reports of human embryonic germ cell derivation( _- c9 J% i6 ?* E
& I4 ?9 e2 m# n" yPreparation of Human PGCs for Culture
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% [' C8 G; _: O( u5 o: QLiu et al. . 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.( e& w7 x3 W: h+ ?# ?* c! z0 ]
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Basic In Vitro Culture Media
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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%¨C15% fetal bovine serum (FBS), ESC-tested FBS, or knockout serum replacement (KO-SR) (Table 1).
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2 O% G/ u+ a# c: bWhereas 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 . 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.
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Feeder Layers
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The reporting groups have predominantly used either mouse STO fibroblasts or primary mouse embryonic fibroblasts (pMEFs) as feeder layers (Table 1). Shamblott et al. . Similar to this experience, it is conceivable that different cell types, providing differing factor combinations, will improve the sequential derivation and maintenance of hEGCs.1 r0 L' Z- ^3 a. B' i: c* W9 I7 ^; J
: v) U3 p$ L6 x1 T# gMedia Additives and Critical Factors$ d* \- {) g2 c! l
7 [$ F( l% k/ u. WIn 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.* ?$ k- o2 g6 f+ e7 X6 W- R' V% C
b0 O3 e5 N0 DStem 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 (our unpublished data).7 H$ y( m# T1 y, Y6 L& x. E( M, g( y3 T
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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 .
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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 ., R% p! n' f' E& p* H; K+ b
. W/ s6 R/ M& s' ?In PGCs, however, the effects of LIF are debatable. LIFR/ gp130/Stat3b signaling inhibits the progression to meiosis of cultured female mPGCs (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).
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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 .
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! J1 n' a' a8 I( D7 [1 i! @Fgf2 functions as a potent mitogen in many cell types and induces telomerase activity in cultured mouse neural precursor cells . This suggests overlap or synergism with SCF in maintaining telomerase activity of mPGCs when taken into culture.. u7 P/ n( U7 }& [5 _
: ~$ C4 ]# g. e8 {Forskolin. All groups deriving hEGCs have included forskolin, which raises intracellular cAMP levels and stimulates mitosis in cultured mPGCs .
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' t: u0 r" T7 y QAlthough 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 , the converse effect of GDNF and EGF on mouse or human EGC derivation and maintenance is unknown.) i4 U% `5 a! Z% ^( d
7 _! ]$ r& d' e6 @& v% k0 vGrowth Characteristics of Human PGCs and EGCs
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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." M% G1 L7 z+ `
, Y) x$ H8 B/ FAs 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 . In stark contrast, culturing the ICM benefits from maintained intercellular contact with the same cell type.. I H/ x2 y( z& e
3 ^. E$ Z- q, I9 z. f9 W9 GFigure 3. Behavior of human primordial germ cells (PGCs) and embryonic germ cells (EGCs) in culture. (A): Cultured gonadal explant with limited outgrowth of alkaline phosphatase (AP)-positive PGCs. Strong AP activity is preserved within the explant where contacts with supporting somatic cells are maintained. (B): Colony containing AP cells with a more tightly packed morphology, in contrast to (C), where the cells have adopted a more open, migratory-like morphology, and (D), an open network of vigorously proliferative AP cells that lack obvious colony formation. Bar = 500 µm.
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mESCs and mEGCs are derived from inbred strains, such as 129/Sv mice, which display a high incidence (1%) of spontaneous teratocarcinoma and yield mEGCs with relative efficiency ; Fig. 3). 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¡ªtypically 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.
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Several groups, including ourselves, have noted difficulty in maintaining hEGCs undifferentiated long-term . 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.9 {0 X+ z; o0 j4 |# {
, O* L( g6 P" T' B' \! IIn Vitro and In Vivo Characterization of hEGCs
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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 .* v7 N( F3 H! d" D* _
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Gene Expression Analyses
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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 .
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Table 2. Characterization of pluripotency markers in published reports of human embryonic germ cell derivation K$ `7 j' s' s, b
7 n7 o0 w. A0 x1 \7 i9 z2 Y7 oEvidence for Pluripotency of hEGCs: In Vitro and In Vivo1 [$ R4 X! F/ f4 ]* r" K
5 f, {' S6 N( C5 U8 s8 o5 MSelf-renewal of hEGCs has been documented by continuous cultures that retain markers such as OCT4 and SSEAs (Table 2). 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) . These apparently conflicting observations, possibly due to factors intrinsic or extrinsic to hEGCs, or a combination of both, require clarification.
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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 . 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).
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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.
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Therapeutic Potential of Human EGCs
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/ X! ^+ h8 K5 PImportant 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 . Taken together, these studies argue for the inclusion of hEGCs in stem cell research programs.
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Future Directions
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3 ?5 L1 @" I# D/ Q4 Q. f* ~) nNow 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./ j4 e j7 Z! L4 n. k5 E
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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.4 S8 e& b0 O* O2 Q ?/ a3 ] R: D# s
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ACKNOWLEDGMENTS
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+ a @: u! F& g0 y9 k, ^* N5 vWe acknowledge all of the authors of the numerous publications we were unable to cite due to space limitations. L.T. is a U.K. Medical Research Council (MRC) Collaborative Career Development Fellow in Stem Cell Research; R.P. is the recipient of a U.K. MRC Ph.D. Studentship in Stem Cell Research; and N.A.H. is a U.K. Department of Health Clinician Scientist. This work was funded by project grants from the British Heart Foundation (PG/03/021/15128 to D.I.W.) and Hope and the Wellcome Trust (074320 to N.A.H.), the latter award and the fellowship to L.T. generously partnered by the Juvenile Diabetes Research Foundation (www.jdrf.org).
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) ^( n4 f: h+ M8 k0 Z- dDISCLOSURES
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, W5 h9 @% o2 ^/ E/ BThe authors indicate no potential conflicts of interest.; t/ J" N2 G* n# k. i: t# o
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NOTE ADDED IN PROOF7 f5 }; K7 J; o
% I: |: R4 \6 h) l% B- o$ pSince electronic publication of this article, a further group has reported on aspects of the human PGC-EGC lineage and their use in cell therapy.
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