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First published online November 15, 2007
Stem Cells Vol. 26 No. 2 February 2008, pp. 412 -421
doi:10.1634/stemcells.2007-0605; www.StemCells.com
© 2008 AlphaMed Press

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EMBRYONIC STEM CELLS

Expression of Pluripotent Stem Cell Markers in the Human Fetal Testis

Candace L. Kerra, Christine M. Hilla, Paul D. Blumenthalb, John D. Gearharta

aInstitute for Cellular Engineering, Department of Gynecology and Obstetrics, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA;
bDepartment of Gynecology and Obstetrics, Stanford University, Stanford, California, USA

Key Words. Embryonic germ cells • Embryonic stem cells • Human • Pluripotency • Primordial germ cells • Testis

Correspondence: Correspondence: Candace L. Kerr, Ph.D., Institute for Cellular Engineering, Department of Obstetrics and Gynecology, Johns Hopkins University, Broadway Research Building, Suite 771, 733 North Broadway, Baltimore, MD 21205, USA. Telephone: 410-614-3444; Fax: 410-955-7427; e-mail: ckerr{at}jhmi.edu

Received on July 27, 2007; accepted for publication on November 6, 2007.

First published online in STEM CELLS EXPRESS  November 15, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Human primordial germ cells (PGCs) have proven to be a source of pluripotent stem cells called embryonic germ cells (EGCs). However, the developmental potency of these cells in the fetal gonad still remains elusive. Thus, this study provides a comprehensive analysis of pluripotent and germ cell marker expression in human fetal testis 7–15 weeks postfertilization (pF) and compares this expression to their ability to derive EGCs. Although the majority of germ cells expressed stem cell markers stage-specific embryonic antigen (SSEA) 1, SSEA4, EMA-1, and alkaline phosphatase, only a small percentage of those (<1%) expressed OCT4, CKIT, and NANOG. Specifically, the number of OCT4+/CKIT+/NANOG+ cells significantly increased in the developing cords during weeks 7–9, followed by a gradual decline into week 15 pF. By week 15 pF, the remaining OCT4+/CKIT+/NANOG+ cells were found in the cords surrounding the periphery of the testis, and the predominant germ cells, CKIT+ cells, no longer expressed OCT4 or NANOG. Based on morphology and early germ cell marker expression, including VASA, PUM2, and DAZL, we suggest these cells are mitotically active gonocytes or prespermatogonia. Importantly, the number of OCT4+ cells correlated with an increase in the number of EGC colonies derived in culture. Interestingly, two pluripotent markers, Tra-1–60 and Tra-1–81, although highly expressed in EGCs, were not expressed by PGCs in the gonad. Together, these results suggest that PGCs maintain expression of pluripotent stem cell markers during and after sexual differentiation of the gonad, albeit in very low numbers.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
During development, pluripotent cells progress through sequential differentiation pathways to generate all cell types in the body. The ability to study these cells, called stem cells, in culture provide model systems to study early human embryonic development as well as providing potential therapeutic strategies for treating degenerative diseases. Currently, pluripotent stem cells have been derived from only two sources, embryonic and germ cells (reviewed in [1, 2]). Embryonic stem cells (ESCs) are derived from the inner cell mass of preimplantation blastocysts [3–5], whereas embryonic germ cells (EGCs) and embryonal carcinoma cells (ECCs) are derived at later stages of development from primordial germ cells (PGCs). EGCs are derived from karyotypically normal, diploid PGCs isolated from last stage gastrulation to early gonadal development in the fetus [1, 6]. In contrast, ECCs are PGC-derived stem cells isolated from adult testicular tumors called teratocarcinomas (mixed germ cell tumors) that form within the seminiferous tubules of the male gonads [7–9]. Collectively, all of these cell types share the general properties of pluripotent stem cells. They exhibit unlimited self-renewal, and they can give rise to derivatives of all three embryonic germ layers, as demonstrated by (1) experimentally induced teratocarcinomas, (2) the wide variety of cell types found in embryoid bodies (EBs) and cell culture, and/or (3) experiments in chimeric mice, including germ-line transmission.

Although there are currently more than 50 reports worldwide on the successful derivation of ESCs [10], only a handful of laboratories have reported on EGCs [11–14]. Therefore, one critical factor to optimizing EGC derivation is in part dependent on the ability to identify and characterize their founder population in the fetal gonad. To derive human EGCs, gonadal tissue is mechanically and enzymatically dispersed, and cells are cultured with mitotically inactivated mouse Sandoz inbred Swiss mouse (S or SIM); 6-thioguanine resistant; ouabain resistant (STO) cells [15]. The question remains whether EGCs are derived from colonies of pluripotent PGCs or are an artifact of culturing a unipotent population of germ cells into a pluripotent state. Indeed, EGC derivation is accomplished between 7 and 12 weeks postfertilization (pF), at a time when PGCs have already begun to differentiate into prespermatogonium in the gonad and the gonad itself is also undergoing sexual differentiation into a committed testis [16, 17].

Although human PGCs have been studied for many decades, the nature of their pluripotent potential is still under considerably debate. Historically, PGCs were considered unipotent in that they appeared to be short-lived in vivo and in vitro and were unable to contribute to chimeras or form embryoid bodies in culture [18]. However, this perception has now been challenged by several studies that have independently shown the expression of OCT4, CKIT, and NANOG in a small number of cells in the human gonad at various stages of prenatal development. These data provide evidence for the presence of pluripotent PGCs, which persist in utero and which give rise to EGCs in culture and ECCs in vivo. If so, this would provide a unique relationship to compare the attributes of PGCs and EGCs for studying mechanisms of pluripotency, a relationship that cannot be declared for ESCs and their progenitors, which are composed of a heterogeneous population of cells of the inner cell mass. However, these analyses would require an enrichment of PGCs that, based on their small numbers and slow replication rates, would depend on sorting techniques using cell surface markers. Indeed, other markers of pluripotency have been identified as present in the inner cell mass of the blastocyst and shared by ESCs, ECCs, and EGCs. These include cell surface proteins CKIT, stage-specific embryonic antigens (SSEAs) SSEA1, EMA1, SSEA3, and SSEA4, and tumor rejection antigens (TRA) 1–60 and 1–81 (TRA-1–60, TRA-1–81). However, unlike OCT4 and NANOG, these markers are not exclusive to stems cells, making the significance of these markers unclear in these cells or in PGCs. Although these markers are readily used to study EGCs, there is a paucity of information regarding the relationship of their expression with OCT4 and NANOG expression in PGCs in vivo. There are also conflicting reports on the expression of SSEA1 in the gonad. Although several reports demonstrate similar patterns of SSEA1 staining in human gonads at different ages, some groups have identified these cells as germ cells, whereas others describe them as Sertoli cells, both using various morphological criteria to identify the cells. Sertoli cells are somatic cells that support germ cells in their development and as such wrap long extensions with cell-cell adhesion around germ cells. Inherently, these contacts make it nearly impossible for immunostaining to distinguish between these two populations for localization of a cell surface antigen such as SSEA1. For this reason, in the following report, SSEA1-positive cells were isolated, shown to develop into EGCs, and characterized by immunostaining and reverse transcription-polymerase chain reaction (RT-PCR) with germ cell and Sertoli cell markers. Cell types were distinguished using early germ cell-restricted markers including VASA and PUM and markers of Sertoli cells such as anti-Müllerian hormone, vimentin, and intermediate filament protein Cytokeratin-18 (CK-18). Together, the data from this study characterized the expression of known markers of pluripotency in the early gonad by performing comprehensive, immunohistochemical, and RNA analyses prior to and just following testicular differentiation. Using this approach, populations of PGCs were characterized and the progression of their differentiation was followed during the first and second trimesters.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Collection of Tissue
Gonadal tissues were obtained using a protocol approved by the Joint Committee on Clinical Investigation of the Johns Hopkins University School of Medicine from human fetuses 7–15 week postfertilization as a result of termination of pregnancy. Gestational age was estimated through a comparison of anatomical markers, including crown heel and crown rump measurements, limb and digit formation, and the first day of the last maternal menstrual cycle. Ages are discussed in terms of fetal development and not the age from the last menstrual period. After acquisition, all tissue was immediately prepared for either cryopreservation or paraffin embedding.

PGC Isolation and Embryonic Germ Cell Derivation
Primordial germ cells were isolated using magnetic cell sorting (MACS) technology using an indirect labeling of cells with magnetically tagged goat anti-mouse IgM antibodies toward a mouse anti-SSEA1 antibody. Briefly, gonads were minced in 1 mg/ml collagenase and incubated at 37°C for 20 minutes, rinsed, and incubated with 1:5 antibody for 30 minutes on ice. Afterward, secondary antibody was applied at a 1:100 dilution for another 30 minutes on ice. After a final rinse, cells were applied on magnetic columns and sorted off the column in culture medium per the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com). PGCs were grown in culture, and EGC derivation was propagated as described previously [11] on {gamma}-irradiated STO feeder layers.

Cryostaining
Fresh tissue was rinsed in Dulbecco's phosphate-buffered saline (DPBS), frozen in O.C.T. freezing compound (Sakura Finetek Inc., Torrance, CA, http://www.sakura-americas.com) Tissue-Tek and stored at 80°C. For immunohistochemistry, tissue were cut into 5-µm sections, placed on slides (ProbeOn Plus; Fisher Scientific International, Hampton, NH, http://www.fisherscientific.com), and immediately prepared for indirect immunofluorescent staining. Sections were fixed either in 4°C acetone for 10 minutes to detect cell surface markers or in 4% paraformaldehyde for 10 minutes to detect nuclear markers. Antibodies and the concentrations used are summarized in supplemental online Table 1. Briefly, cell surface antibodies were diluted in 15% goat serum in DPBS and incubated on sections for 1 hour at room temperature, whereas nuclear antibodies were diluted in 5% goat serum and incubated overnight at 4°C. All antibodies were detected by using fluorescently labeled goat anti-mouse secondary antibodies (1:200 dilution; Molecular Probes, Eugene, OR, http://probes.invitrogen.com) in 15% goat serum in DPBS for 1 hour at room temperature. Sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) and mounted using ProLong antifade mounting medium (Molecular Probes). Sections stained with a nuclear and cell surface antibody followed the protocol for nuclear staining. Negative controls were also performed on each gonad, including incubations with secondary antibodies only and with mouse ascites fluid for monoclonal antibodies. Negative controls for polyclonal antibodies against NANOG and VASA consisted of preincubating tissue with goat and rabbit prebleed serum, respectively, prior to the addition of secondary antibodies.

Paraffin Staining
For paraffin embedding, gonadal tissue was fixed in 4% paraformaldehyde overnight at 4°C. The next day, the tissue was washed three times in DPBS for 15 minutes at 4°C and then dehydrated at 4°C for 15 minutes in 50% ethanol followed by two washes in 70% ethanol and embedded using standard procedures. Sections were cut 5 µm thick, mounted on slides (ProbeOn Plus; Fisher Scientific), baked overnight at 50°C, and stored at 4°C. For staining, sections were then deparaffinized and rehydrated through a series of graded alcohols at room temperature. For antigen retrieval, tissue sections were brought to a boil in 0.01 M citrate buffer (pH 6.0) for 2 minutes and then cooled for 3 minutes, a process that was repeated four times in fresh buffer. Sections were then incubated in 2% H2O2 in phosphate-buffered saline (PBS) for 10 minutes at room temperature to quench endogenous peroxidase activity, incubated in 1% bovine serum albumin (A4378; Sigma-Aldrich) in DPBS for 1 hour at room temperature to block endogenous biotin, and then placed in either 15% horse serum or 15% rabbit serum containing 1% bovine serum albumin for 30 minutes at room temperature to block nonspecific binding. For immunohistochemical staining, sections were incubated overnight at 4°C in a sealed, humidified chamber with primary antibodies (supplemental online Table 1). Antibody binding was visualized using the Elite Vectastain ABC kit (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) and Sigma-Aldrich Fast 3,3'Diaminobenzidine tablets (D4293; Sigma-Aldrich) per the manufacturers' instructions. Sections were counterstained with 1% methyl green, dehydrated through a series of graded alcohols and xylene, and mounted with Permount (SP15100; Fisher Scientific). Negative controls were performed by excluding primary antibodies. Double stainings were performed by using a combination of the same detection methods but with different substrates: Fast Blue/naphthol ASMX phosphate (F3378 and N5000; Sigma-Aldrich) for blue staining and 3-amino-9-ethylcarbazole/N,N-Dimethylformamide (A5754 and D4254; Sigma-Aldrich)/H2O2 for red staining, without counterstaining. Alkaline phosphatase staining was performed on paraffin-embedded tissue by fixing sections in 4% formaldehyde for 15 minutes followed by an incubation in 0.4 mg/ml sodium {alpha}-naphthyl phosphate, 1 mg/ml Fast Red TR salt (Sigma-Aldrich), and 4 mM MgCl2 for 20 minutes at room temperature.

Immunocytochemistry
After MACS sorting, SSEA+1 and SSEA–1 cells were plated on STO feeder layers. For immunocharacterization, cells were fixed for 10 minutes in either 4% paraformaldehyde (PFA) for cell surface antigens or 4% PFA in 0.3% Triton X for cytoplasmic and nuclear antigens. After three washes, cells were blocked in 10% bovine serum albumin (BSA) for 30 minutes. Primary antibodies (supplemental online Table 1) were then added at a 1:50 dilution for 1 hour at room temperature. After three 5-minute washes in PBS, horseradish peroxidase-conjugated secondary antibodies in 10% BSA were added for another hour. Antibody binding was visualized using the Elite Vectastain ABC kit (Vector Laboratories) and Sigma-Aldrich Fast 3,3'Diaminobenzidine tablets (D4293; Sigma-Aldrich) per the manufacturers' instructions.

Fluorescent In Situ Hybridization
The sex of each gonad was determined using CEP X (SpectrumOrange)/CEP Y (SpectrumGreen) DNA probes (Vysis, Downers Grove, IL, http://www.vysis.com). Slides were prepared as described above, fixed in Carnoy's fixative for 45 seconds, pretreated in 1 M sodium thiocyanate for 5 minutes at 75°C, and postfixed in 100% methanol for 1 minute. Sections were then denatured in 60% formamide + 2 x standard saline citrate (SSC) (sodium chloride and citric acid) buffer, pH 5.3, at 75°C for 3 minutes, followed by 1 minute in cold 70%, 95%, and 100% ethanol, and then incubated with CEP X/Y DNA probe overnight at 37°C. The following day, sections underwent three posthybridization washes in 60% formamide (Sigma-Aldrich) + 0.3% Nonidet P40 (Igepal; Sigma-Aldrich) + 2 x SSC, pH 5.3. Sections were then counterstained with DAPI and mounted using ProLong antifade mounting medium (Molecular Probes).

Microscopic Imaging
Fluorescent images were captured at magnifications of x40–x400 magnification using a Nikon Eclipse E800 microscope (Nikon, Melville, NY, http://www.nikon.com) equipped with x4–x40 Plan Apo lens. Alexa Fluor 594, Alexa Fluor 568, and CEP X SpectrumOrange probe fluorescence was detected using a G2ERHOD 541,551-nm excitation filter, a 575-nm dichroic mirror, and a barrier filter with a bandwidth of 590. Alexa Fluor 488 and CEP Y SpectrumGreen probe was detected using a fluorescein isothiocyanate excitation filter, a 505-nm dichroic mirror, and a barrier filter with a bandwidth of 515,555 nm. DAPI magnifying was detected using a standard DAPI/Hoechst filter set, UV 2E/C 340,380-nm excitation filter, 400-nm dichroic mirror, and a barrier filter with a bandwidth of 435,485 nm. Barrier filters were manufactured by Chroma ATE, Inc. (Irvine, CA, http://www.chromaate.com). Images were captured with a Photometrics 20 MHz cooled interlined charge-coupled device camera and imported into Metamorph software, version 6.2 (Molecular Devices, Sunnyvale, CA, http://www.moleculardevices.com).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Stem cell marker expression was observed in 70 testes between 7 and 15 weeks pF using the antibodies summarized in supplemental online Table 1. Fluorescence in situ hybridization (FISH) using X/Y probes was used to confirm sex of the tissue. The results are shown in the supplemental online data, demonstrating a FISH produced from male gonads showing green staining of the Y chromosome and red staining of the X chromosome in the nuclei of cells compared with X chromosome staining alone in the female (supplemental online Fig. 1). Figure 1A summarizes the coexpression of pluripotent markers SSEA1 and SSEA4. Significant staining for both markers was detected in the primary sex cords compared with controls (supplemental online Fig. 2). Formation of the testicular cord became apparent in 7-week pF testes showing SSEA1 (green) and SSEA4 (red) colocalization (yellow). By 8 weeks pF, the basal lamina was more developed around the primary sex cords, making them more distinct, and by 9 weeks pF, the cords, as seen by SSEA1 and SSEA4 staining, became elongated and branched out radially within the medulla. Although results showed that most cells expressing SSEA1 were positive for SSEA4, occasionally cells that demonstrated significantly more intense SSEA1 staining than SSEA4 could be seen in the cords; the significance of these cells is uncertain but warrants future investigation. SSEA4 expression also occurred in somatic cells surrounding the cords (arrows), as well as in the rete region and occasionally in the cord. SSEA4 expression around the cords suggests that this expression originates from mesenchymal cells, which have been reported to develop around the cords and differentiate into peritubular or myoid cells during this time period [19]. Thus, based on their morphology and location, the formation of elongated SSEA4+ cells surrounding the cords by 13 weeks pF suggests developing peritubular cells. EMA1 and SSEA1 staining was indistinguishable (data not shown). Most notable was that many more cells expressed SSEA1 and SSEA4 in the cords compared with those expressing OCT4 and CKIT (Fig. 1B). Since SSEA1 and SSEA4 is expressed in a number of cell types in the developing fetus, colocalization studies were performed to compare OCT4 coexpression in these cells. Results showed that all OCT4+ cells consistently expressed SSEA1 and SSEA4 (Fig. 2). When the numbers of SSEA1+ cells were compared at 7 weeks pF, less than 1% of these cells also expressed OCT4. SSEA1+, OCT+ cells were found in the testis but declined significantly during the time period tested, up to 15 weeks pF. Rounded morphology and the large nucleus-to-cytoplasm ratio in the OCT4+ cells were consistent with PGC morphology.


Figure 1
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  		Figure 1. Expression of SSEA1, SSEA4, or OCT4, and CKIT in male fetal gonad. (A): Indirect fluorescent labeling of SSEA1 alone, SSEA4 alone, and combined male gonads 7, 9, and 13 wk pF. Colocalization of SSEA1 (green) and SSEA4 (red) is demonstrated in the testicular cords (yellow). SSEA4 staining alone is also seen in the r region and in the peritubular cells (arrow) surrounding the Cs by 13 wk pF. BM is indicated by arrowheads. (B): Indirect fluorescent labeling of OCT4 and CKIT expression in male gonads 7, 9, and 13 wk pF. Increase in OCT4 expression is demonstrated in the cords by wk 7 pF followed by a subsequent decrease after 9 wk pF, whereas CKIT expression localized in the cord increased over this period of time. 4,6-Diamidino-2-phenylindole (blue) was used as a nuclear stain. Scale bar = 30 µm. (C): Correlation between the number of CKIT+ cells counted per microscopic field (magnification, x200) and the number of OCT4+ cells counted at the same age. Mean ± SD; n = 3; p < .05. Abbreviations: BM, basement membrane; C, testicular cord; pF, postfertilization; r, rete; SSEA, stage-specific embryonic antigen; wk, weeks.

 


Figure 2
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  		Figure 2. Co-expression of OCT4 with SSEA1 and SSEA4 in fetal gonad. Indirect fluorescent labeling in male gonad ~10 weeks postfertilization demonstrating coexpression of OCT4 (green) and SSEA1 (red) (A) and OCT4 (green) and SSEA4 (red) (B). 4,6-Diamidino-2-phenylindole stain (blue) was used as a marker for cell nuclei. Abbreviation: SSEA, stage-specific embryonic antigen.

 
From the time course study, it was also apparent that at 7 weeks pF, there were twice as many CKIT-expressing cells as OCT4-expressing cells, whereas by 8 weeks, similar numbers of cells that stained CKIT and OCT4 were seen on adjacent sections from the same gonad (Fig. 1C). Whether this was a result of CKIT+, OCT4+ cell proliferation or continued CKIT+, OCT4+ cell migration into the gonad warrants further investigation. However, by 9 weeks pF, the number of OCT4+ cells appeared to decrease substantially by 15 weeks pF, whereas the number of CKIT+ cells increased during the weeks investigated. Colocalization studies show that most but not all the cells that expressed OCT4 also expressed CKIT, whereas only a few CKIT+ cells expressed OCT4. However, when OCT4 expression was compared with that of another pluripotent marker, Nanog (Fig. 3), coexpression studies revealed that most OCT4+ cells also expressed NANOG, whereas all NANOG-expressing cells expressed OCT4. Taken together, these data suggest that differentiation occurs from an OCT4+, NANOG+, CKIT+ cell population that over the course of only a few weeks decreases its OCT4+, NANOG+ expression and retains CKIT. By 9 weeks pF, OCT4+ cells also expressed VASA, an early germ cell marker. Specificity of antibodies against CKIT, OCT4, NANOG, and VASA was demonstrated by the lack of staining in negative controls (supplemental online Fig. 2).


Figure 3
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  		Figure 3. Co-expression of OCT4 with NANOG, CKIT, and VASA in fetal gonad. Indirect fluorescence labeling of OCT4 (green) in the cords of a ~9-week postfertilization fetal testis showing colocalization with NANOG (red) (A) and CKIT (red) (B). Cells were found that were OCT4+, NANOG– (arrow) and OCT4+, NANOG+ (yellow) (A) and OCT4+, CKIT+ (yellow) and CKIT+ alone (B). (C): Colocalization of OCT4+ cells and VASA+ (yellow) confirmed germ cell origin in these cells. 4,6-Diamidino-2-phenylindole (blue) was used as a nuclear stain.

 
Together, these data provide circumstantial evidence that pluripotent cells reside in the gonad during EGC derivation. However, since EGCs are acquired from mincing whole gonadal tissue and because they require strict culturing conditions to form, the nature of the cells from which they are derived is still questionable. Therefore, it was not known to what extent, if any, the cells were pluripotent. To address this question, we compared the relationship between the number of OCT4+ cells present in the gonad with the efficiency of deriving EGC colonies. The results showed that the number of OCT4+ cells correlated with the number of embryonic germ cell colonies that were formed in vitro between age-matched specimens, suggesting that the initial population of cells founding EGC colonies may come from an OCT4+ PGC population and not from dedifferentiated germ cells (Fig. 4). The dramatic increase in the number of OCT4+ cells between 7 and 8 weeks pF suggests that either PGC migration into the gonad is still occurring during this time or there is an increase in PGC amplification. EGCs were not derived from testis older than 13 weeks pF, consistent with previous reports [11–13, 20].


Figure 4
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  		Figure 4. Effects of age on OCT4 expression in the fetal gonad and ability to derive embryonic germ cells in culture. Correlation between the age of the testis in weeks postfertilization and the efficiency of deriving new embryonic germ cell colonies ({circ}) and the number of OCT4-positive cells counted per microscopic field at the same age ({diamond}). Mean ± SD; n ≥ 2; {chi}2 = 0.05. The increase in the efficiency of EGC derivation occurred concomitant with an increase in the number of OCT4+ cells found in the fetal testis. Abbreviation: EGC, embryonic germ cell.

 
One important aspect of EGC derivation is the ability to isolate germ cells from the gonad. Based on the immunostaining presented here, an efficient method for obtaining these cells would be through using the cell surface marker SSEA1. However, two cell types are present in the sex cords of the developing testis, germ cells and the somatic, Sertoli cells, and it has been previously reported that the germ cell to Sertoli cell ratio is ~1:15 in the fetal testis during this period [21]. In the present study, staining of SSEA1 and SSEA4 appears to occur in a majority of the cells in the primary sex cords, some of which also express OCT4, NANOG, and CKIT. To assert whether SSEA1+ SSEA4+ OCT– cells are germ cell or Sertoli cells, colocalization studies with either SSEA1 or SSEA4 were performed using antibodies against the Sertoli cell-specific antigen anti-Müllerian hormone (AMH) and the germ-cell restricted markers VASA and PUMILIO-2. These results from our study demonstrated staining of AMH in areas inside the cords during the first trimester; these areas do not appear to overlap SSEA1. In contrast, significant overlap can be seen between SSEA1 and three germ cell markers, VASA, alkaline phosphatase, and PUMILIO-2 (Fig. 5). Furthermore, cells expressing AMH alone (arrow) can clearly be distinguished outside the cords, representing migratory pre-Sertoli cells. Nonetheless, given the limitations of immunostaining and the close relationship between Sertoli cells and germ cells, it can be difficult to distinguish between these two populations of cells in the cords. For this reason, SSEA1+ cells were isolated with an SSEA1 antibody using magnetic cell sorting to further characterize cell lineage. After sorting, SSEA1+ cells grown in culture expressed the germ cell marker alkaline phosphatase and produced EGC colonies after 1 week [22] (Fig. 6). In comparison, the SSEA1– populations were mostly unable to produce EGCs, except for a single colony formed by one experiment, which we attribute to the loss of a progenitor from antibody capture.


Figure 5
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  		Figure 5. Sertoli and germ cell marker expression in the male gonad. Comparison of indirect fluorescence labeling of stage-specific embryonic antigen 1 (SSEA1) (green) staining in a fetal testis between 9 and 11 weeks postfertilization with the Sertoli cell marker AMH (red) at magnifications of x200 (A) x400 (B). SSEA1 and AMH were expressed in different cells. AMH+ cells were found in the cord and in migratory pre-Sertoli cells (arrows). In comparison, SSEA1 demonstrated colocalized expression (yellow) with the germ cell marker VASA (red) at magnifications of x200 (C) x400 (D). Similar pattern of SSEA1 expression (green) also occurred with AP (red, AP alone; yellow, with SSEA1 overlap) (E) and in paraffin with SSEA1 (red) and PUMILIO-2 (blue) showing colocalization in the cords (brown) (F). DAPI (blue) was used as a nuclear stain in (A–E). Abbreviations: AMH, anti-Müllerian hormone; AP, alkaline phosphatase; DAPI, 4,6-diamidino-2-phenylindole.

 


Figure 6
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  		Figure 6. Increased efficiency of embryonic germ cell deviation from sorted SSEA1+ cells. Alkaline phosphatase staining of primordial germ cells (PGCs) (arrows) isolated from an 8.5-week-old gonad 24 hours after magnetic cell sorting with SSEA1 and plated on mitotically inactivated STO. (A): PGCs without sorting. (B): Purified PGCs after sorting. (C): An EGC colony produced from unsorted cells exposed to SSEA1 antibody alone. (D): EGC colonies produced after sorting (magnification, x100). (E): SSEA1– cells did not express alkaline phosphatase and rarely produced an EGC colony after culturing on feeders. The rare instance of a EGC colony from an SSEA1– population is attributed to cells that escaped antibody capture. Abbreviations: EGC, embryonic germ cell; SSEA, stage-specific embryonic antigen.

 
Although these data confirmed that at least a subset of SSEA1+, SSEA4+ cells are of a germ cell origin, they did not prove whether SSEA1 and SSEA4 are also expressed by Sertoli cells. For this purpose, RT-PCR was used on SSEA1+ cells to determine whether they also expressed markers for AMH, CK-18, and vimentin. Results showed that SSEA1+ cells, although they express early germ cell markers Dazl, Pumilio, and Vasa, do not express detectable levels of markers expressed by Sertoli cells, including AMH, CK-18, and vimentin (data not shown). Furthermore, when protein expression of these markers was compared between SSEA1+ and SSEA1– cells, AMH expression was shown only in the SSEA1– cell population, whereas only SSEA1+ cells expressed VASA and PUM-2 (supplemental online Fig. 3).

Interestingly, the stem cell markers TRA-1–60 and TRA-1–81 showed intense staining in the lining of the mesonephric ducts but could not be detected in the gonad at any age studied (Fig. 7). These results suggest that the expressions of these two markers are induced in culture, proposing a possible association between their expression and the pluripotentiality of embryonic germ cells in culture. Collectively, these data suggest that primordial germ cells initially express SSEA1, SSEA4, OCT4, NANOG, alkaline phosphatase (AP), and CKIT and that during early differentiation in the gonad, prior to their mitotic inactivation, they gradually lose OCT4, AP, and NANOG expression while retaining expression of CKIT and VASA.


Figure 7
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  		Figure 7. Expression of TRA-1–60 and TRA-1–81 was not detected in the gonad. Indirect fluorescence labeling of TRA-1–60 (A, C, E) and TRA-1–81 (B, D, F) expression in a first-trimester (6.5 weeks postfertilization [pF]) (A, B) and second-trimester (13 weeks pF) (C–F) fetal testis. TRA-1–60 or TRA-1–81 staining was not detected in these gonads or in any from the first or early second trimester studied. However, TRA-1–60 (E) and TRA-1–81 (F) expression was found at all ages studied by the cells lining the mesonephric ducts (arrows) as well as in the secretions inside the ducts. 4,6-Diamidino-2-phenylindole (blue) was used as a nuclear stain. Abbreviation: T, testis.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
This study characterized the progression of germ cell development in early human embryogenesis. Specifically, the expression of markers associated with pluripotency was compared during a period of time in which PGCs are able to form pluripotent cell lines. EGCs have been derived from the human fetal gonad from 5 to 12 weeks pF [11–14] and in a comparable developmental stage in mice suspected to produce progenitors of ECCs [8]. Whereas other studies have reported on a few pluripotent markers in the human fetal gonad, this study performed a comprehensive evaluation of human germ cells in early development by comparative expression of more than a dozen markers associated with early germ cell and stem cell identity. This was accomplished using a combinatorial approach looking at biochemical and immunohistochemical characterization of cells in the early gonad, as well as those isolated by the stem cell marker SSEA1. Importantly, this report demonstrates the relationship between multiple markers shown to collectively bind to identical germ cells in vivo. Although many of these markers are also expressed by differentiated cell types, we were able to compare their expression with OCT4 and NANOG expression in vivo and show evidence demonstrating correlative expression with EGC derivation in vitro.

In human embryos, ~100 primordial germ cells appear between the third and fourth week of gestation in the endoderm of the dorsal wall of the yolk sac, near the allantois. PGCs then proceed to migrate through the hindgut during the 4th week and the dorsal mesentery in the 5th week, when ~1,000 PGCs reach the genital ridge ([23, 24]). Once PGCs enter the gonadal ridge they are generally referred to as gonocytes. In fact, the term gonocyte was originally used in rodents to distinguish the postmigratory PGCs from those in the gonad based on morphological distinctions alone [25, 26]. By 7 weeks pF, sexual differentiation of the male gonadal ridge into a testis begins [16, 17], followed by germ cell expansion from approximately 3,000 in week 6 to approximately 30,000 by week 9 pF [21]. However, one issue that remains is the level of heterogeneity of the germ cells present during the first and second trimesters of development. For instance, in mouse, fetal germ cells appear to be a homogenous population in the developing testes, demonstrating a uniform appearance, AP activity [27–29], OCT4 expression [30, 31], SSEA1 and SSEA4 expression [32], and EMA1 expression [33]. However, several reports have shown, based on morphological and biochemical criteria, that the germ cell population at this time in humans is heterogeneous and includes unique populations that express Oct4, Nanog, or c-Kit at various time points [34–37]. When we compared the expression among these markers during early germ cell fate, we were able to observe what appeared to be a progressive development in their expression patterns. In the earliest specimens studied (6.5–7 weeks pF), similar numbers of germ cells expressed OCT4+, NANOG+, and CKIT+ and demonstrated a consistent germ cell-like morphology with a large nucleus and prominent nucleolus. In addition, we were able to observe the coexpression of these markers in the same cells, which demonstrated that after ~8 weeks pF, OCT4 expression was not always coincident with that of NANOG and CKIT. In fact, OCT4, NANOG, and CKIT expression appeared first in the earliest gonads examined (6.5 weeks pF), although by 9 weeks pF OCT4 and NANOG staining decreased, whereas the number of CKIT+ cells increased in the cords. As such, these cells provide a model to examine the relationship between the expression of these three markers in pluripotency. Indeed, there is evidence for the regulation of Nanog and Oct4 on each other's gene expression [38], as well as mutations in CKIT that affect expression of these markers in ovarian dysgerminomas [39]. Furthermore, differences in expression between OCT4 and CKIT may begin to address distinctions describing different populations of germ cells during the first trimester. For example, a distinction between OCT4+, CKIT+ PGCs and OCT4– CKIT+ gonocytes may be drawn, and the significance of their ability to form EGCs or ECCs warrants further investigation.

In addition to studying the expression of these pluripotent transcription factors, this study also compared the expression of cell surface markers associated with pluripotency, SSEA1 and SSEA4. Here, we report for the first time the coexpression of SSEA1 and SSEA4 on the fetal testis, which identified identical germ cells in the gonad, whereas SSEA4 alone was also expressed in the rete lining and in the cells that support the cords. Individually compared, SSEA1 and SSEA4 expression was also found in all OCT4- and NANOG-positive cells. However, as shown in this study, during differentiation, SSEA1+, SSEA4+ gonocytes and prespermatogonial cells develop that lose OCT4 and NANOG expression but gain expression of PUMILIO-2 and VASA [40, 41]. Gaskell et al. [35] reported three types of early germ cells that they identified as gonocytes (OCT4+/C-KIT+/MAGE-A4–), intermediate germ cells (OCT4low/–/C-KIT–/MAGE-A4–), and prespermatogonia (OCT4–/C-KIT–/MAGE-A4+). In this report, we have identified an OCT4–, NANOG–, CKIT+ population that expresses SSEA1 and, based on EGC derivation studies, may also be capable of deriving EGCs. Future studies are warranted that will be able to separate the SSEA1+, OCT4– population from the OCT4+ population to determine their pluripotentiality.

Studies involving human embryonic germ cell derivation have also shown SSEA1+ staining in the gonad, suggesting that these are the founding population for EGCs [13]. However, others have reported similar patterns of staining in the human fetal testis using markers for Sertoli cell expression, including AMH, also called Müllerian inhibiting substance [42, 43]. AMH is produced by immature Sertoli cells and is responsible for the regression of Müllerian ducts in the male fetus. The original attempts by Bendsen et al. [21, 44] to distinguish Sertoli cells from germ cells reported more Sertoli cells than germ cells during this period. In these studies, cell types were distinguished solely on the morphology of the nuclei. Sertoli cells had an elongated nuclei, whereas germ cells were more rounded.

In our studies, hematoxylin and eosin staining of paraffinized tissues demonstrated tightly compacted, rounded cells in tubular formation throughout the first trimester, such that it was not possible to distinguish cellular phenotypes based on morphology alone. In addition to our colocalization studies comparing AMH and SSEA1, we further distinguished between the two cell types by comparing marker expression after sorting populations based on SSEA1 expression. After MACS sorting, VASA, PUM, and AP expression was demonstrated only in the SSEA1+ cells, whereas AMH expression was found only in the SSEA1– population. Furthermore, an examination of microarray analyses of SSEA1+ purified cells showed that this population of cells expressed early germ cell markers, such as Dazl, Pumilio2, and Vasa, and did not express the Sertoli-cell markers Amh, vimentin, or Ck18. Although there is no dispute that AMH-secreting Sertoli cell progenitors are in the cord at this stage, our study distinguishes this cell type from SSEA1+ SSEA4+ germ cells during this period in development.

Interestingly, TRA-1–60 and TRA-1–81 were not detected in the gonad at any age studied, even though staining of the antigens was strongly detected in the lumen of the mesonephric ducts. Although there is no other report of TRA-1–81 expression in the fetal testis, one group has reported on the expression of TRA-1–60. However, in their reports, TRA-1–60 staining was not consistent in all first trimester testis studied and was observed in only 2 of 11 male gonads from the second and third trimesters [45, 46]. However, their report of increased expression of TRA-1–60 in testicular carcinoma patients makes it interesting to study further the role of TRAs in conversion of PGCs into EGCs and ECCs.

Taken together, our data have demonstrated that there is a small population of OCT4+, NANOG+, CKIT+ cells in the developing gonad that, although having the potential to be the founders for pluripotent stem cells in culture, do not express the TRA-1–60 and TRA-1–81 antigens, which are expressed by EGCs and ECCs. Furthermore, this study has provided a number of interesting findings regarding the development and differentiation of human germ cells in the fetal testis, including a very small population of PGCs with a molecular signature expressing OCT4, NANOG, CKIT, SSEA1, SSEA4, and alkaline phosphatase. Being able to identify the molecular signature of PGCs will provide valuable tools for isolating this distinct population of germ cells for cytological and molecular analysis. Of great interest is the period of development in germ cell fate studied here since it is the time at which human germ cells demonstrate the ability to form EGCs and ECCs. This information will be critical in future studies for identifying the progenitor cells of EGCs and ECCs, as well as the mechanisms involved in their conversion.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
We thank Drs. Ann Burke, Rameet Singh, Roxanne Jamshidi, and Ann Lawler, as well as the Birth Defects Laboratory at University of Washington, for assistance in acquiring tissue. We also thank Joyce Axelman for unwavering support and assistance in cell culture. Their enthusiasm and patience throughout this endeavor was greatly appreciated. This work was graciously supported by the Institute for Cellular Engineering, Johns Hopkins University, Baltimore, MD.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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