Stem Cells http://www.peprotech.com/
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jung, J. G.
Right arrow Articles by Han, J. Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jung, J. G.
Right arrow Articles by Han, J. Y.

Stem Cells 2005;23:689-698 www.StemCells.com
© 2005 AlphaMed Press

Development of Novel Markers for the Characterization of Chicken Primordial Germ Cells

Jin Gyoung Junga,b, Duk Kyung Kima, Tae Sub Parka, Seon Duk Leeb, Jeong Mook Lima, Jae Yong Hana

a Department of Food and Animal Biotechnology, Seoul National University, Seoul, Korea;
b Avicore Biotechnology Institute Inc., Gyeonggi-Do, Korea

Key Words. Chicken • Primordial germ cell • Characterization • Stage-specific embryonic antigens • Lectin • Integrin

Correspondence: Jae Y. Han, Ph.D., Division of Animal Genetic Engineering, School of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea. Telephone: 822-880-4810; Fax: 822-874-4811; e-mail: jaehan{at}snu.ac.kr


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study was undertaken to develop novel markers for chicken primordial germ cells (PGCs), which are of potentially enormous value in transgenic research. Gonadal cells collected from 5.5-day-old chicken embryos were cultured in a Dulbecco’s minimal essential medium and the PGC colonies formed during the primary culture period were subcultured three times. Characterization of the PGCs with the candidate marker reagents was performed on the mixed cell population 2 hours after seeding, after the primary culture period (day 10), and after the third passage (day 40). Mouse embryonic stem (ES) cells were used as controls. The cytochemical reagents investigated included periodic acid-Schiff (PAS) stain, antibodies to stage-specific embryonic antigens (SSEA-1, SSEA-3, and SSEA-4), antibody to epithelial membrane antigen (EMA)-1, antibodies to integrins {alpha}6 and ß1, several lectins (Solanum tuberosum agglutinin [STA], Dolichos biflorus agglutinin [DBA], concanavalin A agglutinin [ConA], and wheat germ agglutinin [WGA]), and double staining with antibodies to SSEA-1, SSEA-3, SSEA-4, integrin {alpha}6, or integrin ß1 and then with the lectin STA. Densitometric quantification was used to identify PGC-specific markers. The results showed that chicken PGCs were stained selectively by PAS and by antibodies to SSEA-1, SSEA-3, SSEA-4, EMA-1, integrin {alpha}6, and integrin ß1. The control mouse ES cells reacted with PAS, anti-SSEA-1, and anti-EMA-1 antibodies, as well as with antibodies to integrins {alpha}6 and ß1, but not with antibodies to SSEA-3 and SSEA-4. Chicken PGCs reacted with the lectins STA and DBA, but mouse ES cells reacted with STA and WGA. The results of double staining of PGC colonies subcultured three times showed that the intensity of staining was not altered by concomitant use of the marker reagents. This study demonstrated that, in addition to PAS and antibodies to SSEA-1 and EMA-1, new specific markers of chicken PGCs are recognized by the lectins STA and DBA and by antibodies to SSEA-3 and SSEA-4 and inte-grins {alpha}6 and ß1. Double staining using these newly developed markers might be the method of choice for rapid characterization of chicken PGCs.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Owing to their physiological and developmental characteristics, birds have tremendous value in transgenic research. The production of transgenic birds, and transgenic chickens in particular, through germline transmission is now considered to be the most efficient strategy for the production of animal bioreactors. The simple composition of the egg proteins ensures the feasibility of the chicken as a bioreactor. Furthermore, the chicken is an ideal animal model for diverse types of neurological research. The size of the chicken egg permits ease of manipulation in vitro, and recent advances make it possible to produce germline chimeras by transfer of primordial germ cells (PGCs) into heterogeneous embryos [1–7]. The feasibility of avian transgenic systems has led to progress toward establishment of the required infrastructures and reagents. Much effort has been devoted recently to the development of novel gene-targeting methods for chicken PGCs.

The characterization of cells maintained in vitro is essential for the success of pluripotent cell research. In chicken germ cell research, staining with periodic acid-Schiff (PAS) and antibodies to stage-specific embryonic antigen (SSEA)-1 and epithelial membrane antigen (EMA)-1 have been used for the detection of specific markers of PGCs, and the pluripotency of candidate PGCs has been confirmed by induction of germline transmission via the transfer of cells into recipient embryos [8]. Such methods are somewhat insufficient to fully characterize chicken PGCs, and it is occasionally considered that SSEA-1 and PAS-positive cells are presumptive PGCs. Apparently, additional effort to identify novel markers is urgently required for further development of avian transgenic systems, and this study was designed to identify and develop novel markers for the characterization of chicken PGCs. The candidate markers tested in this study were selected based on previous research, and the staining sensitivity was monitored both qualitatively and quantitatively to distinguish PGC-specific markers.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
General Information
All procedures for animal management, reproduction, and surgery were performed in accordance with the standard protocols of Seoul National University. The Institutional Review Board of the Department of Animal Science and Technology, Seoul National University, approved the research proposal and the relevant experimental procedures. All chickens were maintained at the University Animal Farm, College of Agriculture and Life Science, Seoul National University.

Experimental Design
The characterization of the markers present on PGCs was conducted using PGCs within 2 hours after seeding (immediately after plating down), PGCs collected after the primary passage in vitro (day 10 of culture, after colony formation), or PGCs collected after the third passage (day 40 of culture). Mouse embryonic stem (ES) cells were used as controls, except in the double-staining experiments. The intensity of the histochemical or immunocytochemical staining for marker candidates on the PGCs or ES cells was subsequently examined. In experiment 1, PAS staining alone was used. In experiment 2, immunostaining with antibodies against SSEA-1, SSEA-3, SSEA-4, and EMA-1 was investigated. Experiment 3 examined immunostaining with antibodies against integrin {alpha}6 and integrin ß1, and experiment 4 examined cytochemical staining with the lectins Solanum tuberosum agglutinin (STA), Dolichos biflorus agglutinin (DBA), wheat germ agglutinin (WGA), and concanavalin A agglutinin (ConA). In experiment 5, double staining of PGCs (collected after the third passage in vitro) with the lectin STA in combination with antibodies to SSEA-1, SSEA-3, SSEA-4, integrin {alpha}6, or integrin ß1 was evaluated.

In experiments 1 through 4, the reactivities of candidate markers on PGCs were monitored both quantitatively and qualitatively, whereas only qualitative analysis was performed in experiment 5. For the qualitative analysis, both fluorescent microscopy and light microscopy were used. For quantification of the specificity of the markers, the intensities of staining of PGC colonies and feeder cells were determined by densitometry, and the differences were compared.

Collection and Culture of Chicken PGCs and Mouse ES Cells
Stage-28 (5.5-day-old) White Leghorn chicken (Gallus gallus domesticus) embryos were collected and rinsed with Ca2+- and Mg2+-free phosphate-buffered saline (PBS) to remove residual yolk. The gonads were removed via a medial section of the abdomen with sharp tweezers and dissection under a stereomicroscope. The gonadal cells, which included the PGCs, were dissociated from the embryonic gonads by gentle pipetting in 0.05% (vol/vol) trypsin solution supplemented with 0.53 mM EDTA and subsequently centrifuged at 200g for 5 minutes. According to our standard procedure [8], 1 x 106 gonadal cells were seeded onto each 100-mm plastic culture dish. Dulbecco’s minimal essential medium (DMEM) (Gibco Invitrogen, Grand Island, NY) supplemented with 10% (vol/vol) fetal bovine serum (FBS) (Hyclone, Logan, UT), 2% (vol/vol) chicken serum (Gibco Invitrogen), 1 x antibiotic-antimycotic supplement (Gibco Invitrogen), 10 mM nonessential amino acids (Gibco Invitrogen), 10 mM Hepes (Gibco Invitrogen), 0.55 mM ß-mercaptoethanol (Gibco Invitrogen), 2 ng/ml human leukemia inhibitory factor (LIF) (Sigma-Aldrich Corp., St. Louis), 0.5 ng/ml human basic fibroblast growth factor (Sigma-Aldrich), 3 ng/ml stem cell factor (Sigma-Aldrich), and 10 ng/ml human insulin-like growth factor-1 (Sigma-Aldrich) was used for PGC culture. As our preliminary data showed that approximately 1% of the seeded gonadal cells were PGCs, the number of PGCs seeded initially was approximately 104 cells per dish. The seeded cells were then cultured in a CO2 incubator maintained at 37.5°C in an atmosphere of 5% CO2 in air with 60%–70% relative humidity.

From the mixed cell population that was initially seeded, the gonadal stromal cells formed a monolayer within 5 days after culture, whereas the PGCs formed colonies by 7 days of culture. On day 10, the colony-forming PGCs were passaged, and the cultures were subsequently passaged three times at intervals of 10 days.

Mouse ES cells (E14 cell line of 129 strain; ATCC, Manassas, VA) were used as controls for the characterization of candidate markers. For culture of the ES cells, a feeder cell layer was established by growing 2 x 105 STO cells in each well of a six-well plate. When the cells reached 80% confluence, they were inactivated for 2 hours with 10 µg/ml mitomycin-C (Sigma-Aldrich). Mouse ES cells were thawed, seeded (2 x 105 per well) onto the STO feeder cells, and cultured with DMEM supplemented with 15% (vol/vol) FBS, 1.7 mM L-glutamine (Gibco Invitrogen), 0.1 mM ß-mercaptoethanol, 1 x antibiotic-antimycotic supplement, and 2 ng/ml LIF. For immunostaining, 1 x 104 ES cells were seeded onto one well of a 24-well plate containing an inactivated feeder layer of STO cells.

Periodic Acid-Schiff Staining
In experiment 1, isolated PGCs were collected before seeding, colony-forming PGCs were collected after one or three passages (10 or 40 days) in culture, and cultured mouse ES cells were fixed in 50 mM phosphate buffer containing 2% (vol/wt) glutaraldehyde, 2% (vol/vol) formaldehyde, and 2 mM MgCl2 for 10 minutes. After rinsing in PBS, the cells were then immersed in periodic acid solution (Sigma-Aldrich) for 5 minutes and subsequently treated with Schiff’s reagent (Sigma-Aldrich) for 15 minutes. All procedures were performed at room temperature, and the stained PGCs were observed under an inverted microscope (TE2000-U, Nikon, Tokyo).

Reagents for Immunocytochemical and Histochemical Analysis
Mouse anti-SSEA-1 monoclonal immunoglobulin M (IgM) antibody (MC-480), mouse anti-SSEA-3 monoclonal IgM antibody (MC-631), mouse anti-SSEA-4 monoclonal IgG antibody (MC-813-70), and mouse anti-EMA-1 monoclonal IgM antibody were purchased from the Developmental Studies Hybridoma Bank (Iowa City, IA). Mouse anti-chicken integrin ß1 monoclonal IgG antibody was purchased from Sigma-Aldrich, whereas mouse anti-chicken integrin {alpha}6 monoclonal IgG antibody was from Chemicon International Inc. (Temecula, CA). For detection of the primary antibodies, either rhodamine-conjugated goat anti-mouse IgG and IgM (H+L) antibodies (Jackson Laboratories Inc., Bar Harbor, ME) or alkaline phosphatase-conjugated antibodies and detection kit (Dako Universal LSAB2 kit; Dakocytomation, Carpinteria, CA) were used. Four types of fluorescein isothiocyanate (FITC)–conjugated lectins, STA, DBA, WGA, and ConA, were purchased from Sigma-Aldrich.

Immunocytochemical Analysis with a Single Antibody or Lectin
In experiments 2 and 3, the alkaline-phosphatase detection system was used for the immunocytochemical analyses using anti-SSEA-1, anti-SSEA-3, anti-SSEA-4, anti-EMA-1, anti-integrin {alpha}6, and anti-integrin ß1 antibodies. Briefly, chicken PGCs or mouse ES cells were fixed in 50 mM phosphate buffer containing 2% (vol/wt) glutaraldehyde, 2% (vol/vol) formaldehyde, and 2 mM MgCl2 for 10 minutes. To minimize nonspecific binding of antibodies, the fixed cells were treated for 30 minutes with 5% (vol/vol) goat serum before immunostaining. The optimal concentration of each antibody was selected based on the results of preliminary experiments (40 µg/ml for anti-SSEA-1 and anti-EMA-1 antibodies and 20 µg/ml for all others). After treatment for 1 hour with the primary antibodies, the cells were sequentially reacted for 10 minutes each with biotinylated anti-mouse immunoglobulins, alkaline phosphatase-conjugated streptavidin, and the substrate chromogen. Endogenous alkaline phosphatase activity in the PGCs was inhibited by treatment with 0.02 M levamisole to limit false-positive results.

In experiment 4, the fixed cells were incubated with FITC-conjugated STA, DBA, WGA, or ConA for 1 hour and then washed three times in PBS. In a preliminary study, the optimal concentrations of the conjugated lectins without nonspecific binding were in the range of 20–60 µg/ml; concentrations in this range were used for the analyses of PGC markers.

Double Immunofluorescent and Lectin Staining
Based on the results of experiments 1 through 4, double staining of PGCs was carried out with anti-SSEA-1, anti-SSEA-3, anti-SSEA-4, anti-integrin {alpha}6, or anti-integrin ß1 antibodies in combination with STA. After fixation, the PGCs were incubated with the primary antibodies for one of the five candidate markers and subsequently treated with rhodamine-labeled anti-mouse IgG or anti-mouse IgM secondary antibodies. After washing several times with PBS, the PGCs were treated with FITC-conjugated STA for 1 hour. The stained PGCs were observed under an inverted fluorescence microscope (TE-300, Nikon).

Densitometric Analysis for Quantification
A densitometer (LAS-3000, Fujifilm, Stanford, CA) for measuring image scanning and a MultiGauge software program (version 2.0, Fujifilm) for analyzing signal intensity were used for quantification of the reaction of the PGCs with each marker reagent. Photographs were taken after immunocytochemical staining, and five different areas with images of both colonized cells and background (feeder layer) were randomly selected on each photograph. The colony and the background in each area occasionally had different staining intensity with tested substrates. The difference in the intensity of staining between the PGCs in the colonies and the background cells was then measured, and the average of the five areas was taken as one replicate. The feasibility of this quantification method was confirmed by the preliminary results; densitometric difference between colony and background was measured in all colonies of one picture, and no different aspects were detected compared with the results obtained from the method suggested. More than three replicates were made for quantification of differential staining and were submitted for statistical analysis.

Statistical Analysis
The quantitative densitometric data were subjected to analysis of variance and the least-square method using the general linear model (PROC-GLM) of the SAS program (SAS Institute, Cary, NC). Probability values less than .05 were considered statistically significant, and the tested substrates were considered to be PGC-specific markers only when both parameters yielded significant differences.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Reactivity of PGCs with Single Marker Candidates (Experiments 1 Through 4)
At 2 hours after seeding, PGCs plated down and were sporadically found in the mixed population of gonadal cells. These cells subsequently formed well-delineated colonies by 7 days of primary culture. The PGC colonies formed in the primary culture were maintained without any observable morphological differentiation until the third passage. Both freshly isolated and colony-derived PGCs stained strongly with PAS (experiment 1; Fig. 1Go). There were several isolated PGCs or small colonies of PGCs, but those all also reacted with PAS (Figs. 1D, 1FGo). As shown in Table 1Go, densitometric quantification also showed a significant difference in the intensity of staining between the PGC colonies and the feeder layers (p < .0003). Mouse ES cells also stained strongly with PAS (p < .05).



View larger version (147K):
[in this window]
[in a new window]
  		Figure 1. Morphology of chicken primordial germ cells (PGCs) cultured for different periods. Gonadal cells containing mixed populations of PGCs and stromal cells were collected from 5.5-day-old chicken embryonic gonads. The colony-forming PGCs from the primary culture were passaged three times. Observations were made on (A, B, C) day 0 (2 hours after seeding), (D, E, F) day 10 (at the end of primary culture), and (G, H, I) day 40 (at the end of the third passage). PGCs were characterized by periodic acid-Schiff (PAS) staining and anti-EMA-1 antibody immunostaining. (J, K, L): Mouse embryonic stem(ES)cells(E14celllineof129strain) were used as controls. PGCs (A, B, and C; arrowheads) were scattered in the mixed gonadal cell population immediately after seeding but subsequently formed well-delineated PGC colonies by the end of the primary culture period. (E, H): The colonies were maintained without any observed morphological differentiation and strongly reacted with PAS stain through the third passage in vitro. Before the formation of colonies during primary culture, the gonadal stromal cells formed a monolayer (GM) of feeder cells. (K): Mouse ES cells were also strongly stained with PAS. To know EMA-1+ pattern in the chicken PGCs and mouse ES cells, immunocytochemical analysis was performed. In consequence, PGCs were positively stained with anti-EMA-1 antibody to PGCs on (C) day 0, (F) primary cultured PGCs, and (I) third-passaged PGCs. (L): Mouse ES cells were also positively stained with anti-EMA-1 antibody. Scale bar = 50 µm. Abbreviation: PC, PGC colony

 

View this table:
[in this window]
[in a new window]
  		Table 1. Binding activitya of colony-forming PGCs on days 10 and 40 of culture and mouse ES cells with marker candidates
 
Immunostaining demonstrated statistically significant (p < .0001) differences in the intensity of staining of chicken PGC colonies and the feeder layers after staining with anti-SSEA-1, anti-SSEA-3, anti-SSEA-4, and anti-EMA-1 antibodies, as shown by both light microscopy and densitometry (experiment 2; Fig. 2Go, Table 1Go). In contrast, mouse ES cells were stained specifically only with anti-SSEA-1 and anti-EMA-1 antibodies, and no significant differences from background were found after staining with anti-SSEA-3 and anti-SSEA-4 antibodies (p > .05). The gonadal stromal cells and the STO monolayers showed no reactivity with any of the anti-SSEA and the anti-EMA-1 antibodies.



View larger version (114K):
[in this window]
[in a new window]
  		Figure 2. Immunocytochemical characterization of colony-forming chicken primordial germ cells (PGCs) on (A, B, C) day 0 (2 hours after seeding), (D, E, F) day 10 (at the end primary culture), and (G, H, I) day 40 (at the end of the third passage). Gonadal cells containing mixed populations of PGCs and stromal cells were collected from 5.5-day-old chicken embryonic gonads. The colonies of PGCs forming on the stromal cell monolayer during the primary culture were passaged three times. (J, K, L): Gonadal stromal cells collected on the same day of observation, and (M, N, O) mouse embryonic stem (ES) cells (E14 cell line of 129 strain) were used as the control groups for characterization of the markers. Antibodies to (A, D, G, J, M) SSEA-1, (B, E, H, K, N) SSEA-3, and (C, F, I, L, O) SSEA-4 were used to characterize the PGCs. Colony-forming PGCs reacted positively with all the tested antibodies, and this reactivity was not influenced by repeated passage. In contrast, no reactivity was detected in the gonadal stromal cell monolayer. Mouse ES cells reacted positively only with anti-SSEA-1 antibody. Scale bar = 50 µm.

 
In experiment 3, PGCs reacted specifically with anti-integrin {alpha}6 and anti-integrin ß1 antibodies (Fig. 3Go). Densitometric quantification showed that the intensity of staining with both anti-integrin {alpha}6 (p < .0001) and anti-integrin ß1 (p < .0001) antibodies was significantly different from that of the background cell layer (Table 1Go). Both antibodies also reacted specifically with the mouse ES cells (p < .001).



View larger version (96K):
[in this window]
[in a new window]
  		Figure 3. Characterization of colony-forming chicken primordial germ cells (PGCs) on (A, B) day 0 (2 hours after seeding), (C, D) day 10 (at the end of primary culture), and (E, F) day 40 (at the end of the third passage) by staining with (A, C, E, G) anti-integrin {alpha}6 and (B, D, F, H) anti-integrin ß1 antibodies. (G, H): Mouse embryonic stem (ES) cells were used as controls. Gonadal cells containing mixed populations of PGCs and stromal cells were collected from 5.5-day-old chicken embryonic gonads. The colonies of PGCs forming on the stromal cell monolayer during the primary culture were passaged three times. Regardless of the collection time, the PGCs stained positively with antibodies to integrins {alpha}6 and ß1 with no non-specific staining of the feeder cell layer. Mouse ES cells were also stained with these antibodies. Scale bar = 50 µm.

 
In the case of the lectin assays in experiment 4, significant differences in the intensity of staining of chicken PGCs compared with the background cell layers were detected by densitometric quantitation after staining with FITC-conjugated STA (p < .001) and FITC-conjugated DBA (p < .0002) (Fig. 4Go, Table 1Go). Fluorescence microscopy demonstrated that the PGC colonies collected at the end of the primary culture or after the third passage were strongly stained with both lectins. In the case of DBA, weak staining was detected in the feeder layer of gonadal stromal cells, but a clear difference between the PGC colonies and the feeder layer was observed.



View larger version (82K):
[in this window]
[in a new window]
  		Figure 4. Characterization of colony-forming chicken primordial germ cells (PGCs) on (A, E, I, M) day 0 (2 hours after seeding), (B, F, J, N) day 10 (at the end of primary culture), and (C, G, K, O) day 40 (at the end of the third passage) by staining with (E, F, G, H) fluorescein isothiocyanate–conjugated lectins Solanum tuberosum agglutinin (STA) and (M, N, O, P) Dolichos biflorus agglutinin (DBA). (D, H, L, P): Mouse embryonic stem (ES) cells were used as controls. Gonadal cells containing mixed populations of PGCs and stromal cells were collected from 5.5-day-old chicken embryonic gonads. The colonies of PGCs forming on the stromal cell monolayer during the primary culture were passaged three times. Fluorescence microscopy showed that (E, F, G) STA reacted strongly with colony-forming PGCs and that this staining was maintained until the end of the third passage. (M, N, O): DBA also strongly stained PGCs but weakly stained the gonadal stromal cells. (H, P): Both lectins stained mouse ES cells weakly compared with the intensity of staining of chicken PGCs. Scale bar = 50 µm.

 
As shown in Figure 5Go, nonspecific binding of ConA and WGA to chicken PGCs and feeder cells was detected. Several colonies showed relatively low intensity of staining, and the staining of feeder stroma cells was not uniform for ConA and WGA in most cases. There were no significant differences in staining intensity between the PGC colonies and the feeder layers (p = .1232 for ConA and 0.7003 for WGA). The mouse ES cells were specifically stained with STA and WGA (p < .05), whereas DBA and ConA showed nonspecific staining.



View larger version (100K):
[in this window]
[in a new window]
  		Figure 5. Characterization of colony-forming chicken primordial germ cells (PGCs) on (A, E, I, M) day 0 (2 hours after seeding), (B, F, J, N) day 10 (at the end of primary culture), and (C, G, K, O) day 40 (at the end of the third passage) and of (D, H, L, P) mouse embryonic stem (ES) cells by staining with fluorescein isothiocyanate–conjugated lectins (E, F, G, H) concanavalin A agglutinin (ConA) and (M, N, O, P) wheat germ agglutinin (WGA). Gonadal cells containing mixed populations of PGCs and stromal cells were collected from 5.5-day-old chicken embryonic gonads. The colonies of PGCs forming on the stromal cell monolayer during the primary culture were passaged three times. (D, H, L, P): Mouse ES cells were used as controls. Fluorescence microscopy showed no specific staining of PGCs by either lectin; both the PGCs and the feeder layer were strongly stained with ConA and WGA. In the control, (H) ConA weakly stained the feeder cells (STO) and the mouse ES colonies, whereas (P) WGA strongly stained the ES cells only. Scale bar = 50 µm.

 
Double Staining with STA and Anti-SSEA Antibodies (Experiment 5)
The PGCs were stained with anti-SSEA-1, anti-SSEA-3, anti-SSEA-4, anti-integrin {alpha}6, or anti-integrin ß1 antibodies in combination with STA. As shown in Figure 6Go, the specificity and intensity of binding of each marker reagent remained unchanged by the combined staining protocol; the colonies that primarily reacted with anti-SSEA-1, anti-SSEA-3, anti-SSEA-4, anti-integrin {alpha}6, or anti-integrin ß1 antibodies were strongly stained with subsequent treatment of lectin-STA. There were no differences in the reactivities of the PGC colonies collected after primary culture and after the third passage (supplemental online data; not shown).



View larger version (84K):
[in this window]
[in a new window]
  		Figure 6. Characterization of colony-forming chicken primordial germ cells (PGCs) on day 40 (at the end of the third passage) by double immunostaining with (C) anti-SSEA-1, (F) anti-SSEA-3, (I) anti-SSEA-4, (L) anti-integrin {alpha}6, or (O) anti-integrinß1 antibodies and (B, E, H, K, N) fluorescein isothiocyanate (FITC)–conjugated Solanum tuberosum agglutinin (STA) (the second column). Colony-forming PGCs were primarily reacted with anti-SSEA antibodies or anti-integrin antibodies and then sequentially stained with FITC-conjugated STA. No competitive binding activity between any combinations of two reagents was detected. Scale bar = 25 µm.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We developed a new panel of markers for the characterization of chicken PGCs. The results of immunostaining demonstrated that anti-SSEA-3, anti-SSEA-4, anti-integrin {alpha}6, and anti-integrin ß1 antibodies and STA and DBA bound specifically to chicken PGCs. These reagents could be used to characterize chicken PGCs together with conventional marker reagents such as PAS, anti-SSEA-1, and anti-EMA-1 antibody. We also showed that double staining of PGCs with the newly developed markers was feasible, which might contribute to rapid detection and accurate characterization of chicken PGCs. The newly discovered markers and double staining protocol can also be used for discerning PGCs from other types of cells.

In birds, the PGCs, which are the progenitors of spermatozoa and ova, originate from the epiblast [9] and appear in the germinal crescent area during the early primitive streak stage [10]. PGCs subsequently migrate into the gonadal ridge through the circulatory system and finally differentiate into the germ cells. Recent success in producing germline chimeras by transfer of gonadal PGCs confirmed the hypothesis that PGCs could retain stem cell pluripotency before the initiation of gametogenesis in the embryonic gonads. However, the gonad-derived, PGC-like cells are occasionally considered presumptive PGCs because of the insufficiency of PGC-specific markers. In this study, chicken gonadal PGCs could be detected by more reagents used for identification of both ES cell–specific and germ cell–specific markers in mammalian species, and these results expanded the pool of specific chicken PGC markers.

Although standard procedures for the detection and identification of murine PGCs have been established for some time, only limited information has been available concerning the characterization of chicken PGCs. The results of this study confirmed the applicability of PAS staining and anti-SSEA-1 and anti-EMA-1 immunostaining, which have conventionally been used to detect chicken PGCs. In our preliminary experiments, however, alkaline phosphatase, a classical ES cell marker, was not useful for the detection of chicken PGCs. Also, in contrast to mouse ES cells, chicken PGCs could be immunostained with anti-SSEA-3 and anti-SSEA-4 antibodies. Furthermore, chicken PGCs and mouse ES cells showed different intensities of staining with the fluorescein-conjugated lectins tested in this study. These results suggest that chicken PGCs have different characteristics compared with mammalian ES cells. It is likely that these putative differences are not associated with pluripotency, because our previous results have clearly shown that PGCs are pluripotent for the induction of both somatic and germline chimerism [7, 8]. Physiological and genetic particularities of chicken PGCs, not related to pluripotency, might be responsible for the observed differences.

Because gonadal PGCs collected from 5.5-day-old embryos induced germline transmission [6], discovering new specific markers for chicken PGCs definitely contributed to developing the standard characterization protocol of chicken PGC as a pluripotent cell. It seems that the gonads of 5.5-day-old embryos are the best for the retrieval of sufficient numbers of PGCs for biotechnological manipulation, and thus the cell characterization has mainly been done after gonadal migration. Although blastodermal cells, the origin of PGCs, were characterized with PAS, SSEA-1, EMA-1, and ECMA-7 in previous research [11, 12], further study is necessary for validating candidate-specific markers for detecting premigratory or migratory PGCs. In addition, there were several reports on the characterization of in situ expression of germ cells on several markers at a later stage of embryo development [13, 14].

SSEAs are carbohydrate antigens associated with various core glycolipids and are routinely used for the characterization of pluripotent cells in mammalian species. SSEA-1 is first expressed in the late-cleavage stages of mouse embryos, and undifferentiated mouse ES cells strongly expressed SSEA-1 epitope [15–18]. Anti-SSEA-1 antibody also reacts specifically with mouse embryonic carcinoma (EC) cells, mouse embryonic germ (EG) cells, and human EG cells. Anti-SSEA-1 antibody has also been used for the detection of PGCs in several species [19–22], but human EC cells do not react with anti-SSEA-1 antibody.

In contrast, SSEA-3 (Galß-globoside) and SSEA-4 (sialyl-Galß-globoside) are epitopes localized at the cell surface that are associated with globoseries glycolipids [23]. These antigens are expressed in cleaving mouse embryos, and antibodies raised against these antigens also bind to human EC cells [23, 24]. Antibodies against SSEA-3 and SSEA-4 bind to monkey ES cells and to human ES, EG, and EC cells [25]. SSEA-1 and SSEA-3 were detected at the cell membrane and in the cytoplasm of mouse germ cells [18]. The results of the present study are the first to demonstrate the presence of SSEA-3 and SSEA-4 epitopes on chicken PGCs.

The EMA-1 is a cell-surface glycoprotein, which specifically binds to mouse ES cells. The EMA-1 is also specific to PGCs in birds [26, 27] and has been conventionally used for the detection of chicken pluripotent cells. PGCs of pig [19] and goat [20] and ES cells of mouse [28] were also stained with anti-EMA-1 antibody. Like EMA-1, it was also reported that 2C9 monoclonal antibody was also effective for detecting chicken PGCs [29], and our preliminary data showed that PGCs used in this study strongly reacted with 2C9 (data not shown).

Integrins are heterodimeric transmembrane proteins that mediate interactions between cells and the extracellular matrix. In the mouse, integrin ß1 plays a key role in the migration of PGCs into the embryonic gonads [30]. It was reported that integrins {alpha}6 and ß1 were localized on the plasma membrane of mouse PGCs [31] and strongly expressed on mouse ES and EC cells [32]. Anti-integrin antibodies have been used to bind these specific surface markers to purify mouse spermatogonial cells [33]. De Felici and Dolci [34] demonstrated that integrins {alpha}6 and ß1 were present in the genital ridges of mouse embryos. In our study, mouse ES cells were also positive for both integrins, which was consistent with the previous results. Based on our results, the expression of these integrins on chicken PGCs might be maintained even after migration into the genital ridge.

As shown in Figures 2Go and 3Go, apparent staining intensity of PGCs was detected after immunostaining. However, the densito-metric quantification staining intensity of each colony in a single area became occasionally different. This might result from minimal concentration of staining agents for avoiding nonspecific staining or might be attributable to the presence of heterogeneous germ cells (coexistence of PGCs and other kinds of germ cells). Otherwise, different staining intensity might reflect subtle physiological difference of each PGC. Further experiments might be necessary for interpreting such paradoxical observation. Single-cell culture of subcultured PGCs might be a good strategy for elucidating the fate and the characterization of individual PGCs.

Lectins are carbohydrate-binding proteins that interact with specific sugar residues included in glycoconjugates. Different types of lectins have strict sugar specificities that have been exploited in the study of the structure of cell-surface carbohydrates and the changes in carbohydrate composition that occur during embryonic development. Histochemistry using lectins has been used to detect the changes in distribution and expression of cell-surface carbohydrates related to cellular development and differentiation. Lectins have also been used to identify and compare the origins of glycoconjugates. Several lectins have served as candidates for histochemical lineage markers of embryonic cells, including PGCs [19, 35–39]. In the chicken, the carbohydrate bound by ConA was found to have a significant role in PGC migration in stage-6 through -12 embryos [40], and WGA, ConA, and STA were shown to bind to PGCs in stage-29 embryos and spermatogonia in stage-46 embryos. Lectins have also been shown to bind to Sertoli cells in the gonadal tissue of stage-36 through -46 embryos [41].

The lectin STA is a chitotriose (GlcNAc3)–binding protein derived from potato tubers and contains approximately 50% carbohydrate, comprised of arabinose and galactose. Positive staining of PGCs with STA might suggest the presence of GlcNAc3 on chicken PGCs. Considering that STA can bind to glycoproteins reacting with PAS reagent, STA-positive cells could be expected to react strongly with PAS stain. Our results support this hypothesis. On the other hand, the lectin DBA recognizes terminal N-acetylgalactosamine residues. Unfertilized mouse ova express DBA-binding glycoconjugates. However, the expression of DBA-binding membrane constituent declines during preimplantation development [42], and mouse PGCs are negative for DBA staining [43]. In the present study, DBA staining was specific for chicken PGCs that were cultured in vitro, which suggested that the expression pattern of the DBA-binding carbohydrates differed between chicken and mouse PGCs.

The results of the present study showed nonspecific binding of WGA and ConA to PGCs and gonadal stroma cells that were cultured in vitro after collection from stage-28 embryos. Considering that all previous studies of lectin binding in chickens were conducted with tissues taken directly from chicken embryos in vivo, it is possible that in vitro culture might affect the affinity of PGCs for lectins; that is, the reactivity to lectins of cells in vivo might differ from that of cells cultured in vitro.

The reactivity of chicken PGCs to the specific marker reagents newly developed in this study was not altered by up to three passages in vitro, which might suggest that the physiological characteristics of PGCs were not changed through the third passage in vitro. However, extension of the PGC culture period is absolutely required for the establishment of stable transgenic technology, which is the ultimate goal of our research on avian pluripotent cells. Horiuchi et al. [44] recently reported that the use of chicken LIF, not mouse LIF, contributed to maintaining an undifferentiated state of chicken blastodermal cells. Apparently, it is necessary for further developing the culture regimen for chicken PGCs. Several medium supplements may be necessary either for deleting from formulation or for replacing with more appropriate substrates to maintain cell survival and undifferentiated state.

It is important not only for effectively detecting chicken pluripotent cells from mixed population but also for well maintaining the undifferentiated state of cultured cells. Chicken PGCs have conventionally been isolated by morphological criteria and subsequently been identified with PAS, SSEA-1, and EMA-1 antibodies. The efficiency of PGC collection was continuously optimized by the results of previous studies [7–9]; the efficiency of PGC retrieval improved 45.1 times from the original level [45], and the rate of germline chimera production increased up to 49.7% [7, 8]. In the future, different combinations of double staining or establishment of triple staining will be a good choice for rapid confirmation of the characterization of chicken pluripotent cells. Concomitant staining with PAS and SSEA-1 could be one alternative for such attempts.

In conclusion, the development of new histochemical and immunocytochemical markers specific for chicken PGCs, presented here for the first time, will further contribute to quick and reliable characterization of PGCs in vitro, including those subcultured for extended periods.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This research was supported by a grant (SC14011) from the Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology and BK21 Project, Republic of Korea.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Wentworth BC, Tsai H, Hallett JH et al. Manipulation of avian primordial germ cells and gonadal differentiation. Poult Sci 1989;68:999–1010.[Medline]

  2. Naito M, Tajima A, Yasuda Y et al. Production of germline chimeric chickens, with high transmission rate of donor-derived gametes, produced by transfer of primordial germ cells. Mol Reprod Dev 1994;39:153–161.[CrossRef][Medline]

  3. Chang I, Yoshiki A, Kusakabe M et al. Germ line chimera produced by transfer of cultured chick primordial germ cells. Cell Biol Int 1995;19:569–576.[CrossRef][Medline]

  4. Tagami T, Matsubasra Y, Hanada H et al. Differentiation of female chicken primordial germ cells into spermatozoa in male gonads. Dev Growth Differ 1997;39:267–271.[CrossRef][Medline]

  5. Han JY, Park TS, Hong YH et al. Production of germline chimeras by transfer of chicken gonadal primordial germ cells maintained in vitro for an extended period. Theriogenology 2002;58:1531–1539.[CrossRef][Medline]

  6. Park TS, Jeong DK, Kim JN et al. Improved germline transmission in chicken chimeras produced by transplantation of gonadal primordial germ cells into recipient embryos. Biol Reprod 2003;68:1657–1662.[Abstract/Free Full Text]

  7. Park TS, Hong YH, Kwon SC et al. Birth of germline chimeras by transfer of chicken embryonic germ (EG) cells into recipient embryos. Mol Reprod Dev 2003;65:389–395.[CrossRef][Medline]

  8. Park TS, Han JY. Derivation and characterization of pluripotent embryonic germ cells in chicken. Mol Reprod Dev 2000;56:475–482.[CrossRef][Medline]

  9. Eyal-Giladi H, Ginsburg M, Farbarov A. Avian primordial germ cells are of epiblastic origin. J Embryol Exp Morphol 1981;65:139–147.[Medline]

  10. Ginsburg M, Eyal-Giladi H. Temporal and spatial aspects of the gradual migration of primordial germ cells from the epiblast into the germinal crescent in the avian embryo. J Embryol Exp Morphol 1986;95:53–71.[Medline]

  11. Pain B, Clark ME, Shen M et al. Long-term in vitro culture and characterization of avian embryonic stem cells with multiple morphogenetic potentialities. Development 1996;122:2339–2348.[Abstract]

  12. Etches RJ, Clark ME, Zajchowski L et al. Manipulation of blastodermal cells. Poult Sci 1997;76:1075–1083.[Abstract/Free Full Text]

  13. Watanabe M, Kinutani M, Naito N. Distribution analysis of transferred donor cells in avian blastodermal chimeras. Development 1992;114:331–338.[Abstract]

  14. Shaw DL, Carsience RS, Etches RJ et al. The fate of female donor blastodermal cells in male chimeric chickens. Biochem Cell Biol 1992;70:1218–1229.[Medline]

  15. Gooi HC, Feizi T, Kapadia A et al. Stage-specific embryonic antigen involves apha1 goes to 3 fucosylated type 2 blood group chains. Nature 1981;292:156–158.[CrossRef][Medline]

  16. Kannagi R, Fukuda MN, Hakomori S. A new glycolipid antigen isolated from human erythrocyte membranes reacting with antibodies directed to globo-N-tetraosylceramide (globoside). J Biol Chem 1982;25:4438–4442.

  17. Solter D, Knowles BB. Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-1). Proc Natl Acad Sci U S A 1978;75:5565–5569.[Abstract/Free Full Text]

  18. Marani E, Van Oers JW, Tetteroo PA et al. Stage specific embryonic carbohydrate surface antigens of primordial germ cells in mouse embryos: FAL (SSEA-1) and globoside (SSEA-3). Acta Morphol Neerl Scand 1986;24:103–110.[Medline]

  19. Takagi Y, Talbot NC, Rexroad CE et al. Identification of pig primordial germ cells by immunocytochemistry and lectin binding. Mol Reprod Dev 1997;46:567–580.[CrossRef][Medline]

  20. Kuhholzer B, Baguisi A, Overstrom EW. Long-term culture and characterization of goat primordial germ cell. Theriogenology 2000;53:1071–1079.[CrossRef][Medline]

  21. D’Costa S, Petitte JN. Characterization of stage-specific embryonic antigen-1 (SSEA-1) expression during early development of the turkey embryo. Int J Dev Biol 1999;43:349–356.[Medline]

  22. Karagenc L, Cinnamon Y, Ginsburg M et al. Origin of primordial germ cells in the prestreak chick embryo. Dev Genet 1996;19:290–301.[CrossRef][Medline]

  23. Kannagi R, Levery SB, Ishigami F et al. New globoseries glycosphingol-pids in human teratocarcinoma reactive with the monoclonal antibody directed to a developmentally regulated antigen, stage-specific embryonic antigen 3. J Biol Chem 1983;258:8934–8942.[Abstract/Free Full Text]

  24. Andrews PW. Human embryonal carcinoma cells in culture do not synthesize fibronectin until they differentiate. Int J Cancer 1982;30:567–571.[Medline]

  25. Shamblott MJ, Axelman J, Wang S et al. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc Natl Acad Sci U S A 1998;95:13726–13731.[Abstract/Free Full Text]

  26. Urven LE, Erickson CA, Abbott UK et al. Analysis of germ line development in the chick embryo using an anti-mouse EC cell antibody. Development 1988;103:299–304.[Abstract]

  27. Urven LE, Abbott UK, Ericson CA. Distribution of extracellular matrix in the migratory pathway of avian primordial germ cells. Anat Rec 1989;224:14–21.[CrossRef][Medline]

  28. Toumadie A, Kusumoto K, Parton A et al. Pluripotent differentiation in vitro of murine ES-D3 embryonic stem cells. In Vitro Cell Dev Biol Anim 2003;39:449–453.[CrossRef][Medline]

  29. Maeda S, Ohsako S, Kurohmaru M et al. Analysis for the stage specific antigen of the primordial germ cells in the chick embryos. J Vet Med Sci 1994;56:315–320.[Medline]

  30. Anderson R, Fassler R, Georges-Labousees E et al. Mouse primordial germ cells lacking beta1 integrins enter the germline but fail to migrate normally to the gonads. Development 1999;126:1655–1664.[Abstract]

  31. Zuccotti M, Giorgi RP, Fiorillo E et al. Timing of gene expression and oolemma localization of mouse alpha6 and beta1 integrin subunits during oogenesis. Dev Biol 1998;200:27–34.[CrossRef][Medline]

  32. Hierck BP, Thorsteinsdottir S, Niessen CM et al. Variants of the alpha 6 beta 1 laminin receptor in early murine development: distribution, molecular cloning and chromosomal localization of the mouse integrin alpha6 subunit. Cell Adhes Commun 1993;1:33–53.[Medline]

  33. Shinohara T, Avarbock MR, Brinster RL. Beta1- and alpha6-integrin are surface markers on mouse spermatogonial stem cells. Proc Natl Acad Sci U S A 1999;96:5504–5509.[Abstract/Free Full Text]

  34. De Felici M, Dolci S. In vitro adhesion of mouse fetal germ cells to extra-cellular matrix components. Cell Differ Dev 1989;26:87–96.[CrossRef][Medline]

  35. Bandyopadhyay S, Chakrabarti J, Banerjee S et al. Prenatal exposure to high galactose adversely affects initial gonadal pool of germ cells in rats. Hum Reprod 2003;18:276–282.[Abstract/Free Full Text]

  36. Fazel AR, Schulte BA, Thompson RP et al. Presence of a unique glyco-conjugate on the surface of rat primordial germ cells during migration. Cell Differ 1987;21:199–211.[CrossRef][Medline]

  37. Fazel AR, Schulte BA, Spicer SS. Glycoconjugate unique to migrating primordial germ cells differs with genera. Anat Rec 1990;228:177–184.[CrossRef][Medline]

  38. Nagano R, Sun X, Kurohmaru M et al. Changes in lectin binding patterns of mouse male germ cell (gonocytes) during prespermatogenesis. J Vet Med Sci 1999;61:465–470.[CrossRef][Medline]

  39. Nakamura M, Yoshinaga K, Fujimoto T. Histochemical identification and behavior of quail primordial germ cells injected into chick embryos by the intravascular route. J Exp Zool 1992;261:479–483.[CrossRef][Medline]

  40. Lee H, Karasanyi N, Nagele RG. The role of the cell surface in the migration of primordial germ cells in early chick embryos: effects of concana-valin A. J Embryol Exp Morphol 1987;46:5–20.

  41. Nagano R, Kanai Y, Kurohmaru M et al. Changes in lectin binding patterns of chick primordial germ cells and Sertoli cells during sexual differentiation. J Vet Med Sci 1995;57:623–627.[Medline]

  42. Muramatsu T. Developmentally regulated expression of cell surface carbohydrates during mouse embryogenesis. J Cell Biochem 1988;36:1–14.[CrossRef][Medline]

  43. Hahnel AC, Eddy EM. Cell surface markers of mouse primordial germ cells defined by two monoclonal antibodies. Gamete Res 1986;15:25–34.

  44. Horiuchi H, Tategaki A, Yamashita Y et al. Chicken leukemia inhibitory factor maintains chicken embryonic stem cells in the undifferentiated state. J Biol Chem 2004;279:24514–24520.[Abstract/Free Full Text]

  45. Kim JN, Kim MA, Park TS et al. Enriched gonadal migration of donor-derived gonadal primordial germ cell by immunomagnetic cell sorting in birds. Mol Reprod Dev 2004;68:81–87.[CrossRef][Medline]

Received August 23, 2004; accepted for publication January 3, 2005.



This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
J. G. Jung, Y. M. Lee, T. S. Park, S. H. Park, J. M. Lim, and J. Y. Han
Identification, Culture, and Characterization of Germline Stem Cell-Like Cells in Chicken Testes
Biol Reprod, January 1, 2007; 76(1): 173 - 182.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jung, J. G.
Right arrow Articles by Han, J. Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jung, J. G.
Right arrow Articles by Han, J. Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
STEM CELLS THE ONCOLOGIST CME ALPHAMED PRESS JOURNALS
http://www.epitomics.com