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Stem Cells 2003;21:598-609 www.StemCells.com
© 2003 AlphaMed Press

Derivation of Human Embryonic Germ Cells: An Alternative Source of Pluripotent Stem Cells

Lee Turnpennya, Sarah Brickwooda, Cosma M. Spallutoa, Karen Pipera, Iain T. Cameronb, David I. Wilsona, Neil A. Hanleya

a Division of Human Genetics and
b Maternal, Fetal, and Neonatal Physiology Group, Fetal Origins of Adult Disease Division, University of Southampton, Southampton General Hospital, Southampton, United Kingdom

Key Words. Pluripotent • Embryonic stem • Primordial germ cells • Embryonic germ • Embryoid body • Differentiation

Neil A. Hanley, MB.Ch.B., Ph.D. and Lee Turnpenny, Ph.D., Division of Human Genetics, University of Southampton, Duthie Building (MP 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, lturnpen{at}hgmp.mrc.ac.uk


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Based on evidence suggesting similarities to human embryonic stem cells, human embryonic germ (hEG) cells have been advocated as an alternative pluripotent stem cell resource but have so far received limited attention. To redress this imbalance, human fetal gonads were collected for the isolation and culture of primordial germ cells at 7-9 weeks postconception. We provide evidence for the derivation, culture, and differentiation of hEG cells in vitro. This evidence includes the expression of markers characteristic of pluripotent cells, the retention of normal XX or XY karyotypes, and the demonstration of pluripotency, as suggested by the expression of markers indicative of differentiation along the three germ lineages (ectoderm, mesoderm, and endoderm) and an associated loss of pluripotent markers. In assessing this differentiation, however, we also demonstrate a hitherto unacknowledged overlap in gene expression profiles between undifferentiated and differentiated cell types, highlighting the difficulty in ascribing cell lineage by gene expression analyses. Furthermore, we draw attention to the problems inherent in the management of these cells in prolonged culture, chiefly the difficulty in preventing spontaneous differentiation, which hinders the isolation of pure, undifferentiated clonal lines. While these data advocate the pursuit of pluripotent hEG cell studies with relevance to early human embryonic development, culture limitations carry implications for their potential applicability to ambitious cell replacement therapies.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
During development, pluripotent cells commit down sequential differentiation pathways to generate all the organs of the body. Harnessing the potential of this phenomenon in vitro provides a model system to study early human embryonic development and also to develop novel strategies of organ regeneration and transplantation [1]. Although farsighted, this latter ambition has provoked increased interest in the properties of human pluripotent stem cells [2-5], following the recent derivation of human embryonic stem (hES) [6, 7] and human embryonic germ (hEG) [8] cells.

A number of criteria are applied in defining ES and EG cells, including the capability of prolonged in vitro self-renewal in the undifferentiated state and the retention of pluripotency, i.e., the capacity to form differentiated derivatives of the three embryonic germ layers: ectoderm, mesoderm, and endoderm [9]. Although teratocarcinomas and human embryonal carcinoma (hEC) cells have provided important developmental models, they have a restricted capacity for differentiation, which limits their exploitation. Furthermore, they tend to be aneuploid, thus constituting a transplantation hazard [9]. hES cells have been derived from cells of the inner cell mass of preimplantation blastocysts obtained via clinical in vitro fertilization procedures. These ‘precursor’ cells normally progress in vivo to form the complete embryo, including the diploid primordial germ cells (PGCs). During development, PGCs migrate from the posterior endoderm of the yolk sac via the gut mesentery to populate the developing gonads, where they proliferate and, ultimately, undergo meiosis to yield spermatozoa or ova [10]. When postimplantation fetuses are obtained following first trimester termination of pregnancy, PGCs can be isolated from dissected premeiotic fetal gonads for the derivation of diploid hEG cells [8].

It is imperative that all potential renewable sources of human pluripotent cells are thoroughly investigated in the search for novel transplantation strategies. Preliminary investigation of hES cells has attracted the interest of many researchers, with access becoming available to the wider research community via the U.S. National Institutes of Health Stem Cell Registry (http://escr.nih.gov/). However, hEG cells have so far received limited attention, with evidence for derivation emanating from a single research group [5, 8]; thus, their potential to provide a widely available, alternative resource of stem cells requires validation. We report evidence for the derivation, characterization, and differentiation of hEG cells from multiple fetuses. In assessing differentiation, the molecular phenotypes of the differentiated cells were compared with the starting cell population originally obtained from the fetal gonad. While confirming the pluripotency of hEG cells (as evidenced by their acquisition of gene expression profiles representative of all three germ layers), we also highlight the complexity, and prompt caution, in assigning cell fate by gene expression profile. Furthermore, we draw attention to problems encountered in managing the differentiation status of these cells—findings that could limit their application to the farsighted goals of transplantation medicine.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Embryo Collection, Gonadal Dissection, and Disaggregation
With local research ethics committee approval and written informed consent, following Polkinghorne guidelines [11], human fetuses at 7-9 weeks postconception were collected at termination of pregnancy. We initiated primary cultures of gonad cells from over 60 fetuses. Dissection was carried out using stereomicroscopy, and gonads were washed in Hanks’ balanced salt solution (HBSS) (Sigma Chemical Co.; St. Louis, MO; http://www.sigmaaldrich.com). Gonads were immersed in 0.01% EDTA for 10 minutes, washed in HBSS, then mechanically disaggregated and incubated in a cell dissociation mix, consisting of 0.25% collagenase, 20 U/ml DNase I (both from Sigma), 2% heat-inactivated newborn calf serum (Invitrogen Life Technologies Ltd; Paisley, UK; http://www.invitrogen.com), and 60 µg/ml CaCl2, in HBSS at 37°C for 1-2 hours, with repeated trituration. Cells were washed in HBSS and filtered through sterile gauze prior to plating.

Cell Culture
Mouse STO fibroblasts (American Type Culture Collection CRL-1503) were mitotically inactivated by exposure to 50 Gy of {gamma}-radiation and plated in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Invitrogen). Dissociated PGCs were plated onto this feeder layer in Knockout (KO)-DMEM (Invitrogen), containing either 15% KO serum replacement (KO-SR) (Invitrogen) or ES-cell-tested FBS (PAA Laboratories Ltd.; Pasching, Austria; http://www.paa.at), 1 mM L-glutamine, 0.1 mM ß-mercaptoethanol (both from Sigma), 0.1 mM nonessential amino acids (Invitrogen), and antibiotics as above. To promote their survival, proliferation, and maintenance in the undifferentiated state, PGCs/EG cells were cultured in the presence of 10 µM forskolin (Sigma), 4 ng/ml human recombinant basic fibroblast growth factor (bFGF) (Cell Sciences; Norwood, MA; http://www.cellsciences.com), and 1,000 U/ml human recombinant leukemia inhibitory factor (LIF) (Chemicon International, Inc.; Temecula, CA; http://www.chemicon.com). During the first 14 days, cultures were sacrificed or sampled for characterization (see below) or colonies were isolated by cloning cylinder and disaggregated with 0.25% trypsin/1 mM EDTA (Invitrogen) for 3-5 minutes at 37°C. Cells were passaged onto fresh feeder layers, and samples were taken for additional characterization. Subsequently, selected cultures were replated onto one of several tissue culture surfaces: 0.1% gelatin, collagen, poly-L-lysine, or culture plastic. To promote differentiation, cells were either left to grow overconfluent or taken into suspension culture, accompanied by the withdrawal of LIF, bFGF, and forskolin from the culture medium. To encourage aggregation of cells in suspension, Ca2+ concentration was elevated to 4.5 mM [12]. Developing embryoid bodies were collected for individual culture in untreated round-bottom 96-well plates for periods ranging from 2-21 days. All cultures were maintained in 5% CO2/95% humidity at 37°C.

Characterization of PGCs and EG Cells
Cells or tissues were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS), or 66% acetone/3% formaldehyde. Alkaline phosphatase (AP) activity was detected as described previously [13]. Monoclonal antibodies to stage-specific embryonic antigen (SSEA)-1, SSEA-3, SSEA-4, and epithelial membrane antigen (EMA)-1 were used according to depositors’ instructions at the Developmental Studies Hybridoma Bank. Antibodies to hTERT (monoclonal; Novocastra Laboratories Ltd.; Newcastle upon Tyne, UK; http://www.novocastra.co.uk) and Oct-4 (polyclonal; Geneka Biotechnology Inc.; Montreal, Canada; http://www.geneka.com) were used following antigen unmasking in 0.01 M citrate buffer at 100°C. Detection of immunoreactivity was visualized by reaction with streptavidin-horseradish peroxidase (S-HRP) (Vector; Burlingame, CA; http://www.vectorlabs.com), 3, 3'-diaminobenzidine (DAB) (VWR International Ltd.; Poole, UK; http://www.vwr.com), and H2O2 (Sigma) via biotinylated anti-mouse (SSEA-1, SSEA-4, EMA-1, hTERT), anti-rat (SSEA-3), or anti-rabbit (Oct-4) secondary antibody. Toluidine blue was used for counterstaining. Expression of Oct-4 and hTERT were also analyzed by reverse transcription-polymerase chain reaction (RT-PCR) (see below).

Karyotyping
Proliferating cells were incubated in culture with KaryoMAX Colcemid (Invitrogen) for 3-4 hours then resuspended in hypotonic solution (1:1 0.4% KCl:0.8% C6H5Na3O7; both from VRW) and incubated at 37°C for 20 minutes. Cells were then resuspended in fixative (3:1 methanol:acetic acid) and stored at 4°C for at least 30 minutes. Following washing with fixative, cells were applied to clean glass slides and air dried. Metaphase chromosomes were stained with 4'6-diamidino-2-phenylidole (Vector) and analyzed with a Zeiss Axioplan2 microscope (Carl Zeiss Ltd.; Herts, UK; http://www.zeiss.com).

Immunohistochemistry of Embryoid Bodies
Embryoid bodies (EBs) were collected individually in drops of molten 1.5% low melting point agarose (Sigma) in PBS at 42°C and fixed in 4% PFA or 3:1 methanol:acetic acid/acetone for 2 hours. EB sections were tested for immunoreactivity against a range of specific monoclonal and polyclonal antibodies raised against: muscle-specific actin (MSA) (1:200 dilution); neurofilament (NF)200 (1:50); vimentin (1:100); pan-cytokeratin (CK) (1:50) (all from Novocastra); amylase (1:50) (Sigma); and nestin (1:50). Detection was via biotinylated anti-mouse (for monoclonal) or anti-rabbit (for polyclonal) secondary antibodies, S-HRP and DAB/H2O2 reaction, followed by counterstaining with toluidine blue.

RNA Extraction and RT-PCR
RNA was extracted using TRI Reagent (Sigma) according to the manufacturer’s instructions. Two to three micrograms of DNase I-treated RNA were used for oligo(dT)-primed first-strand cDNA synthesis with SuperScript II reverse transcriptase (RT) (Invitrogen) at 42°C. PCR was subsequently carried out on 2-5 µl of cDNA template (or without reverse transcription, as a control) for 30 to 42 cycles with the following primer pairs: nestin, forward 5'-AGAGGGGAATTCCTGGAG-3', reverse 5'-CTGAG GACCAGGACTCTCTA-3'; pancreas-duodenum homeobox gene (Pdx-1), forward 5'-GGATGAAG TCTACCAAAGCTCACGC-3', reverse 5'-CCAGATCTTG ATGTGTCTCTCGGTC-3'; amylase, forward 5'-GCTGGGC TCAGTATTCCCCAAATAC-3', reverse 5'-GACGACA ATCTCTGACCTGAGTAGC-3'; alpha-fetoprotein (AFP), forward 5'-AGAACCTGTCACAAGCTGTG-3', reverse 5'-GACAGCAAGCTGAGGATGTC-3'; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward 5'-TTAGCAC CCCTGGCCAAGG-3', reverse 5'-CTTACTCCTTGGAGGCCATG-3'; POU transcription factor (Oct-4), forward 5'-CGTGAAGCTGGAGAAGGAGAAGCT-3', reverse 5'-CA AGGGCCGCAGCTTACACATGTT-3'; hTERT, forward 5'-GTGTGCTGCAGCTCCCATTTC-3', reverse 5'-GCTGCGT CTGGGCTGTCC-3'; vimentin, forward 5'-GGGACCTCT ACG AGGAGGAG-3', reverse 5'-CGCATTGTCAACATC CTG TC-3'; enolase, forward 5'-TGACTTCAAGTCGCCT GATGATCCC-3', reverse 5'-TGCGTCCAGCAAAGAT TGCCTTGTC-3'; hepatocyte growth factor (HGF), forward 5'-GCATCAAATGTCAGCCCTGG-3', reverse 5'-CAACG CTGACATGGAATTCC-3'; HNF-3ß, forward 5'-CTACGC CAACATGAACTCCA-3', reverse 5'-GAGGTCCATGATC CACTGGT-3'; CK-19, forward 5'-CTTTTCGCGCGCC CAGCATT-3', reverse 5'-GATCTTCCTGTCCCTCGAGC-3'; XBP-1, forward 5'-GAGTAGCAGCTCAGACTGCC-3', reverse 5'-GTAGACCTCTGGGAGCTCCT-3'; and GLUT1, forward 5'-CGGCGGACCCTGCACCTCATAG-3', reverse 5'-TGGGGCGACTCACACTTGGGAATC-3'.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of Human PGCs and Derived EG Cells In Vitro
High AP activity is a characteristic of pluripotent stem cells, including PGCs, hEG cells, and hES cells [6, 8]. The migration of AP+ PGCs into the human gonadal ridge was apparent by Carnegie stage (CS)15, which is 33 days postconception (dpc) (Fig. 1A-1DGo). PGC number increased sequentially in the enlarging indifferent gonad of both sexes during the embryonic period (<56 days, Fig. 1E-1FGo). Differences arose during the late embryonic period (~CS21/52 dpc), when PGC mitotic arrest had been precipitated in the 46,XY gonad by Sertoli cell differentiation and formation of the sex cords (Fig. 1GGo). In contrast, PGCs within the female gonadal ridge continued to increase in number during the early fetal period (>56 dpc, Fig. 1HGo), prior to meiotic prophase.



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  		Figure 1. Migration of primordial germ cells into human 46,XY and 46,XX gonadal ridges. Transverse sections showing AP staining at sequential stages during human embryogenesis. A-B) NT = neural tube, GR = gonadal ridge. C-D) Gonadal ridge (double-headed black arrows) from A and B, respectively, shown at higher magnification; single-headed white arrows indicate individual PGCs. E-F) White arrows indicate PGCs within the gut mesentery. G-H) Male and female gonads at approximately 8 weeks pc showing organization of AP+ PGCs in the sex cords of the male gonad, in comparison with the undifferentiated ovary. Size bars represent 1 mm (A-B), 50 µm (C-F), and 100 µm (G-H).

 
In addition to AP activity, PGCs are characterized by a range of cell surface markers. These include: glycolipids known as SSEAs [14, 15] (Fig. 2BGo), the glycoproteins TRA-1-60 and TRA-1-81 [16], and EMA-1 [17] (Fig. 2CGo). Germline cells also retain expression of the transcription factor Oct-4, a marker of pluripotent stem and undifferentiated cells [18, 19], and exhibit telomerase activity [20]. Collectively, these markers provide a means of identifying and tracking pluripotent cells and, via decline in their expression, suggest the onset of differentiation.



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  		Figure 2. Isolation and characterization of primordial germ cells and derived EG cells. A) Human fetal gonads dissected at 8 weeks pc following removal of mesonephros. B-C) 5-µm sections of male fetal gonad (approximately 8 weeks pc) showing immunoreactivity of PGCs to monoclonal antibodies against SSEA-1 and EMA-1, respectively (cf., Fig. 1GGo). D) Stationary and migratory PGCs stained for AP activity 2 days following dissociation and plating onto an STO feeder layer. E) AP staining of a colony of PGCs after 8 days on an STO feeder layer. F) Immunoreactivity of a colony of hEG cells to an EMA-1 monoclonal antibody after 12 days in culture. G) VP culture of colonies of AP+ cells, fixed at 14 days in culture, compared with (inset) an isolated, solitary AP+ cell on an STO feeder layer, typical of a PP culture, fixed at 22 days, 13 days after passaging. H) VP culture of AP+, derived hEG cells on tissue culture plastic in the absence of a feeder layer. Size bars represent 1 mm (A), 100 µm (B, C, E, F and H), 25 µm (D, G inset), and 10 mm (G).

 
We have established a tissue collection regimen, which, to date, has yielded over 300 specimens from first trimester surgical termination of pregnancy. Separate primary cultures of gonadal cells were initiated from 62 dissected specimens. Six of these cultures (9.7%) were lost prior to characterization and passaging; thus, 56 cultures (>90%) were analyzed in the course of this study.

Dissected gonads (Fig. 2AGo) were disaggregated, and dissociated cells were plated onto feeder layers of mitotically inactivated mouse STO fibroblasts in culture medium supplemented with LIF, bFGF, and forskolin, according to a previous report [8]. A proportion of cultures were sacrificed or sampled within the first 12-14 days for determining the survival and proliferation of PGCs. Dissociated PGCs retained high levels of AP activity, enabling the visualization of individual fixed PGCs within 48-72 hours of plating with either of two predominant morphologies, previously termed ‘stationary’ and ‘migratory’ (Fig. 2DGo) [8]. Within the first week, almost all cultures tested (42/44, ~95%) contained cells with biochemical and immunocytochemical characteristics of PGCs/pluripotent cells. Of 24 cultures analyzed within the second week (either before or after first passage), 21 (87.5%) still contained surviving cells that retained these characteristics. Thus, by deduction, PGCs failed to survive up to 14 days in approximately one-eighth (12.5%) of cultures.

We observed that early cultures (<14 days) differed in their growth characteristics and complement of surviving AP+ cells and, as such, we were able to assign them arbitrarily to one of two groups: poorly proliferating (PP) or vigorously proliferating (VP). In general, PP cultures demonstrated limited colony growth: only solitary cells or small groups of cells stained positive for AP or other pluripotent markers in sampled or sacrificed cultures. Although, in some tested cultures, AP+ cells survived passaging (Fig. 2GGo, inset), their proliferation could not be provoked, despite trials of different combinations of medium formulations and culture surfaces (see Materials and Methods). These observations suggested that hEG cells could not be derived from PP cultures, whether initiated from gonads dissected from either male or female fetuses. In contrast, VP cultures became markedly apparent after approximately 7 days, by the formation of multiple distinct colonies growing on and among the arrested feeder cells. These colonies stained heavily for AP (>90% of cells per colony) (Fig. 2E and 2GGo) and were immunoreactive for other characteristic markers (Fig. 2FGo). However, in contrast to the previous report of hEG cell derivation [8], we did not detect SSEA-3 immunoreactivity on PGCs and hEG cells, either in culture or within sections of fetal gonad (data not shown).

Collectively, the growth of colonies arisen from PGCs, and their rapid proliferation, strongly support the derivation of hEG cell populations in VP cultures. At least eight of our cultures (>=14%) demonstrated these properties. Growth could be maintained by passaging on to fresh feeder cells or, occasionally, on either gelatin-coated or, surprisingly, noncoated tissue culture plastic in the absence of a feeder layer (four out of eight VP cultures, Fig. 2HGo). Derived hEG cells from both male (three) and female (five) fetuses retained a 46,XY or 46,XX karyotype, respectively (Fig. 3Go). VP cultures demonstrated continuous proliferation and were expanded by passaging (1 in 5 dilution) at approximately 4-5 day intervals. However, the proportion of AP+ cells in VP cultures declined beyond 10-12 passages. We were unable to prevent this apparent loss of pluripotent markers, despite strict maintenance of the culture conditions that enabled derivation and early successful culture, as previously reported [8]. Similarly, given the failure of prolonged culture on a feeder layer, attempts to maintain cultures on different surfaces (gelatin, collagen, plastic, or poly-L-lysine) failed to preserve the expression of pluripotent markers in over 30 prolonged cultures.



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  		Figure 3. Karyotypes of human PGC-derived cells. A) 46,XX; ten passages. B) 46,XY; nine passages.

 
hEG Cells Form EBs with Associated Decline in Expression of Pluripotent Markers
Suspension cultures were established from over 21 cultures (PP or VP). Subsequent cell aggregation was limited in PP suspension cultures, whereas it was apparent that VP suspension cultures yielded multiple, distinct EB structures (Fig. 4AGo) that resembled those described previously in hES cell studies [3, 4, 21]. Alternatively, plates of hEG cells from three VP cultures were allowed to grow overconfluent on gelatin-coated plates, yielding similar EB structures, in an approach similar to that previously described for hES cells [4]. Differentiation of hEG cells was promoted by the withdrawal of LIF, bFGF, and forskolin from the culture medium. Multiple EBs from five VP cultures, derived from either male or female fetuses, were collected for ongoing individual culture in untreated 96-well plates. Hematoxylin and eosin (H&E) staining of EBs revealed cellular heterogeneity and vacuolar formation, supporting the differentiation of hEG cells (Fig. 4C-4D). These observations were accompanied by the decline or disappearance of pluripotent markers (Table 1Go). For example, AP activity could be detected in EBs during early culture (<2 days), strongly suggesting the aggregation of pluripotent hEG cells (Fig. 4BGo), but was not detected in sections of ‘mature’ EBs (>10 days, data not shown). Immunohistochemistry of male fetal gonad tissue revealed the nuclear localization of Oct-4 (Fig. 4EGo) and the nuclear/nucleolar pattern of hTERT [22] (Fig. 4FGo) within PGCs. By RT-PCR, we analyzed the differential expressions of Oct-4 and hTERT. The loss of transcripts in mature EBs strongly implied the onset of differentiation and, hence, the loss of pluripotency (Fig. 4GGo).



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  		Figure 4. Formation and characterization of EBs. A) EBs formed via the aggregation of hEG cells in suspension culture in the absence of LIF, bFGF, and forskolin. B) Staining of EBs for AP activity, 2 days after formation. C-D) 5-µm sections from two EBs stained with H&E. E-F) 5-µm sections of male fetal gonad (approximately 8 weeks pc) showing immunoreactivity of PGCs to antibodies against Oct-4 and hTERT, respectively (cf., Fig. 1GGo). G) RT-PCR comparison between fetal gonad (G) and ‘mature’ EBs for expression of Oct-4 (247 bp) and hTERT (264 bp), with GAPDH (541 bp) shown as the loading control. The upper panel shows results following reverse transcription (+RT); the lower panel shows negative controls lacking reverse transcription (-RT). Lanes juxtaposed from different gels. RT-PCR product identities were verified by sequencing. Size bars represent 1 mm (A), 100 µm (B-D), and 50 µm (E-F).

 

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  		Table 1. Summary of markers analyzed in determining differentiation of human pluripotent PGCs
 
Embryoid Bodies Derived from hEG Cells Express Ectodermal, Mesodermal, and Endodermal Markers
Evidence for the pluripotency of derived hEG cells was obtained by analyses of cells within sectioned EBs for characteristics of the three germ layers formed by gastrulation. In addition to a varied morphology within sections of EBs, we performed immunohistochemical and RT-PCR analyses for a panel of genes indicative of particular cell fates (Fig. 5Go). The expressions of several genes considered markers of ectodermal (e.g., NF200), mesodermal (e.g., MSA), or endodermal (e.g., amylase) lineages were detected. It is recognized that obtaining evidence for endodermal differentiation of pluripotent stem cells is more recalcitrant than with the other germ layers [23]. Nevertheless, among the examples shown in Figure 5GGo, several genes have been used previously to demarcate the differentiation of the endodermal lineage, being expressed in at least one endodermal cell type [3, 5, 8, 21, 24]. In light of the difficulty in precisely ascribing cell lineage, these results were qualified further by comparative analyses with undifferentiated PGCs (Fig. 6Go and Table 1Go).



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  		Figure 5. Evidence for differentiation within EBs. A-F) 5-µm sections of EBs showing immunoreactivity for a panel of antibodies. (A) NF200, (B) vimentin, (C) MSA, (D) pan-CK, (E) amylase, and (F) nestin. Similar results were obtained by immunohistochemistry of multiple EBs for each antibody. Size bars represent 100 µm. G) RT-PCR analyses of EBs, demonstrating expression of: i) nestin (496 bp), Pdx-1 (218 bp), and amylase (490 bp) and ii) AFP (680 bp), XBP-1 (462 bp), HGF (550 bp), and HNF-3ß(199 bp). i and ii are separate gels with intervening lanes removed for image juxtaposition. The upper panel shows results following reverse transcription (+RT); the lower panel shows negative controls lacking reverse transcription (-RT). Reactions were reproduced at least twice. Arrowhead indicates the product for Pdx-1. All product identities were verified by sequencing.

 


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  		Figure 6. Comparative gene expressions in the fetal gonad and EBs. A-F) 5-µm sections of male fetal testis at 8 weeks gestation. A) AP-staining (dark blue) identifies PGCs within the sex cords, (B) NF200, absent in PGCs, (C) vimentin, positive in PGCs and other cells, (D) MSA, absent in PGCs, (E) pan-CK, positive in PGCs, and (F) amylase, positive in PGCs. Size bars represent 100 µm. G) RT-PCR analyses of gene expression within fetal gonad (G) and EBs: Pdx-1 (218 bp), GLUT1 (492 bp), XBP-1 (462 bp), amylase (490 bp), vimentin (200 bp), and enolase (503 bp). Intervening lanes have been removed for image juxtaposition. The upper panel shows results following reverse transcription (+RT); the lower panel shows negative controls lacking reverse transcription (-RT).

 
Expression of ‘Lineage Markers’ within EBs and the Fetal Gonad
Taken together over time, the disappearance and/or decline of expression of pluripotent and undifferentiated cell markers, and the subsequent detection of germ-layer-specific markers, strongly suggest the loss of pluripotency and the onset of differentiation (Figs. 4 and 5GoGo). However, the expressions of key genes with critical roles in differentiated cells should not be assumed to serve as definitive markers of differentiation, without demonstrating absence within the initial pluripotent precursor cell population. Moreover, due to the cellular heterogeneity of early cultures, it is formally possible that marker detection could represent stray descendants of somatic gonad cells, as opposed to differentiated PGC derivatives. Therefore, we compared the molecular phenotypes of EBs with that of the fetal gonad for each gene described in the EB analyses (Fig. 6Go and Table 1Go). This comparison established whether somatic cell types within the gonad expressed the marker in question, providing confidence that its subsequent detection represented evidence for differentiation of cells from the germ cell lineage. The absence of MSA in the fetal gonad, specifically the PGCs (Fig. 6DGo), provides compelling evidence that its subsequent detection in EBs represents mesodermal differentiation. However, another marker of muscle tissue, muscle-specific enolase, used previously as evidence for mesodermal differentiation from hES cells [3], was expressed in fetal gonad tissue (Fig. 6GGo). Similarly, vimentin, advocated as a neuronal (hence, ectodermal) marker [5] was also detected in the fetal gonad, where its expression was apparent in both PGCs and somatic cells (Fig. 6C and 6GGo). In contrast, the neuronal marker, NF200, was not detected within PGCs, providing more convincing evidence of ectodermal differentiation (Fig. 6BGo). Among ‘endodermal’ markers, pan-CK was robustly detected in PGCs (Fig. 6EGo), and expressions of Pdx-1 and the exocrine hormone amylase were also detected in the fetal gonad by RT-PCR and/or immunohistochemistry (Fig. 6F and 6GGo). Such observations illustrate the complexity of defining differentiation by gene expression profile. Nevertheless, endodermal differentiation was reinforced by the combined detection in EBs of AFP, HGF, and HNF-3ß (Fig. 5GGo), which were not detected within PGCs and other cells of the fetal gonad.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The isolation of human pluripotent cells has generated enormous interest as a novel resource with which to investigate aspects of early human development and pursue ambitious strategies of organ regeneration and transplantation [9, 25, 26]. Although much work has been published extolling the potential of hES cells, equivalent reports on hEG cells have, by comparison, been lacking. However, PGC migration and proliferation in the human fetal gonad provide a source of diploid cells theoretically capable of forming derivatives of all three germ layers. In partial redress of this imbalance, we have demonstrated that, despite sex differences during later embryogenesis (e.g., male testicular cord formation), PGC isolation and EG cell derivation and differentiation were possible from either sex between CS20/50 dpc and approximately 9 weeks postconception.

Human PGCs and hEG cells showed abundant AP activity and expressed Oct-4 and characteristic pluripotent cell surface markers, including SSEA-1. However, SSEA-3 was not detected in the fetal gonadal ridge, isolated PGCs, or derived EG cells. SSEA-3 expression has previously been detected in hES [6], hEC [27], and, inconsistently, hEG cells [8]. Whereas SSEA-3 disappears quickly in differentiating hEC and hES cells [28, 29], SSEA-1 is absent from hES and hEC cells but is associated with the onset of their differentiation [29]. Such observations raise questions about the status of SSEA-1+/SSEA-3- hEG cells, potentially suggestive of partial differentiation. Against this, however, we demonstrated that SSEA-1 marks the surface of PGCs in the developing gonad (Fig. 2Go); thus, in conjunction with other pluripotent markers, it is likely to be a reliable marker of undifferentiated PGCs and hEG cells in vitro.

From studies of mouse germ cells, an EG cell has been defined as a PGC that continues to divide in culture beyond the time it would normally cease to do so in vivo [30]. Upon previous isolation, PGCs have been prone to apoptosis and required careful management of cell culture conditions to promote their survival, their conversion to EG cells, and to limit their spontaneous differentiation [31, 32]. Variations in culture routines have been described to encourage these features, carrying implications for the feasibility of large-scale culture of human pluripotent stem cells (Table 2Go) [2-4, 6-8, 21, 33-35]. Typically, PGCs/EG cells and ES cells from a range of mammalian species have been cultured on feeder layers of growth-arrested mouse embryonic or STO fibroblasts [6, 8, 30, 36-39]. In addition to providing a more physiological attachment surface, the feeder layer conditions the culture medium, to which supplementary factors are added. One of these factors, LIF, is known to reversibly inhibit the differentiation of mouse ES cells in vitro [40, 41]. Similarly, bFGF is apparently necessary for PGC survival, the derivation of mouse EG cells from PGCs, and the inhibition of pluripotent stem cell differentiation [42]. Forskolin enhances intracellular cAMP levels and has been shown to promote the proliferation of mouse PGCs in culture [43]. Nevertheless, despite these measures, one-eighth of our cultures lost all detectable AP activity within 14 days, presumably due to early failure of PGC survival. Thereafter, another 73.5% of cultures failed to demonstrate rapid proliferation of AP+ cells, despite surviving passaging beyond 14 days. Thus, observations of these PP cultures suggest either the failure of PGC survival and/or PGC conversion to EG cells.


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  		Table 2. Comparison of culture methods in some recent studies of human pluripotent stem cells
 
In our experience, VP cultures, that is, those yielding very proliferative hEG cells, became apparent after approximately 1 week. At least 14% of cultures demonstrated robust proliferation of cells, which were positive for AP and other characteristic markers, during early culture. These cells persisted longer in culture, survived through more passages, and, subsequently, aggregated more readily to form EBs. Furthermore, it was possible for hEG cells to proliferate occasionally on gelatin-coated or noncoated tissue culture plastic. Although this proliferation of some VP culture hEG cells in the absence of feeder cells was striking, such an observation of pluripotent cell populations is not unprecedented. Particular mouse ES cell lines have demonstrated this capacity, suggesting culture heterogeneity exists among lines derived from different sources [44]. The inevitable heterogeneity among PGCs/EG cells derived from unrelated human fetuses could, therefore, have been a factor in the inconsistency of their properties in culture. However, we were unable to maintain the undifferentiated state of these cells beyond 10-12 passages. Notably, this occurred for cultures despite maintenance of conditions (including those previously reported [8]) that led to our initial derivation and rapid proliferation of cells, suggesting technique was not contributory. Despite the failure of maintaining a pluripotent stock population, VP cultures were ‘productive’ in yielding the evidence for differentiation presented herein. Interestingly, although the formation of EBs in the presence of LIF, bFGF, and forskolin has been reported for hEG cells [8], we found that differentiation by EB formation, in either suspension or overconfluent cultures, was more efficient in the absence of these factors. However, the spontaneous loss of undifferentiated cells in VP cultures currently precludes detailed analyses of the individual effects of LIF, bFGF, and forskolin on hEG cells. Nevertheless, these VP cultures provide compelling evidence for the derivation of hEG cells.

Akin to other pluripotent stem cell resources, the capacity for differentiation into derivatives of all three germ layers is of interest. Although the ultimate test of pluripotency is the production of chimeric organisms, appropriate ethical constraints prevent this in human cells. Albeit unregulated and unpredictable, EB formation recapitulates gastrulation, the crucial event in the third week of human embryonic development that gives rise to ectoderm, mesoderm, and endoderm. This enactment of the human body plan is essential for subsequent organ development and equally critical in vitro for the transition from a human pluripotent stem cell to one with more defined characteristics, which is required for novel transplantation strategies. The demonstrated aggregation of AP+ hEG cells, and the subsequent decline/disappearance of AP and other pluripotent cell markers, provides assurance that the vast majority of differentiation proceeds from pluripotent cells within the EB environment. Downregulation of pluripotent markers is associated with the progression of differentiation [29]. However, direct analysis of such differentiation in fetal-gonad-derived cultures is far from straightforward. The cellular heterogeneity of the starting population is a complicating factor—fetal gonads contain cell types other than PGCs. Pluripotent cells can spontaneously differentiate in culture [7], and the difficulty in managing the differentiation status of hEG cells has been recognized by others [45]. Furthermore, a varying degree of cell death occurs within EBs, as observed for ES cells [7]. In line with other descriptions of either hES or hEG cells, we studied a panel of genes indicative of particular cell lineages. For example, mesodermal differentiation is represented by the expression of MSA, not present within PGCs or hEG cells but expressed in isolated groups of cells within the EBs. NF200 similarly represents the ectodermal lineage. Most markers were inconsistently detected across all the EBs tested, indicative of variable patterns of differentiation occurring within these structures.

Endodermal differentiation in pluripotent stem cells appears more difficult to obtain and characterize [23]. For example, cytokeratin intermediate filament proteins are expressed in cells with endodermal fates, but are also present in skin cells of ectodermal origin [46]. Postgastrulation, Pdx-1 is essential for development of the pancreas from foregut endoderm and necessary for glucose-regulated insulin production in differentiated beta cells [47]. RT-PCR has detected Pdx-1 expression in undifferentiated mouse ES cells [48]. Similarly, we detected Pdx-1 transcripts within human cells isolated from the fetal gonad. Therefore, these data limit the isolated use of Pdx-1 expression as a marker of pancreatic endoderm differentiation from pluripotent stem cells. Likewise, although amylase is characteristically produced by the endodermal structures of the exocrine pancreas and salivary gland, it was also produced by PGCs of the fetal gonad. And XBP-1, a transcription factor with a role in liver development [49], was strongly expressed in both EBs and the fetal gonad. However, more specific to the endodermal lineage, we did detect, in EBs, the fetal liver marker AFP, and also HGF and HNF-3ß, which were absent from PGCs or hEG cells, providing evidence for germ-layer-specific gene expression not seen within the pluripotent precursor population.

Taken together, our results complement and support those of the only other group to report the derivation and pluripotent properties of hEG cells, and whose culture methodology we have followed. Clearly, however, extensive characterization of differentiated cells is essential, necessitating caution in the interpretation of cell phenotypes, as undifferentiated human PGCs and hEG cells express a variety of genes, including several often considered characteristic of postgastrulation cell lineages. Moreover, in our experience, it has so far proven difficult to obtain and maintain an undifferentiated population of these karyotypically normal cells in prolonged culture, despite strict maintenance of the conditions that led to hEG cell derivation from several fetuses. While a tendency to spontaneously differentiate would not exclude hEG cells from important experimentation, it lessens their likelihood of providing unlimited expansion of an in vitro resource of human pluripotent stem cells for ambitious cell replacement therapies. Nevertheless, it remains worthwhile to develop improved and stable culture conditions that will enable the full potential of hEG cells to be realized.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study was funded by the School of Medicine, University of Southampton, and HOPE (Wessex Medical Trust). L.T. is a Juvenile Diabetes Research Foundation (JDRF) Postdoctoral Training Fellow. N.A.H. is a UK Department of Health Clinician Scientist. S.B. and K.P. are also supported by the JDRF. We are grateful to Dr. Rosalia Marzella, Università di Bari, Italy, for karyotype advice and assembly. The antibody to nestin was a kind gift from Dr. Ron McKay. The monoclonal antibodies SSEA-1, SSEA-3, SSEA-4 (developed by D. Solter), and EMA-1 (developed by M. Eddy) were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received April 14, 2003; accepted for publication May 27, 2003.



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