标题: Development of Novel Markers for the Characterization of Chicken Primordial Germ [打印本页] 作者: 江边孤钓 时间: 2009-3-5 10:50 标题: Development of Novel Markers for the Characterization of Chicken Primordial Germ
a Department of Food and Animal Biotechnology, Seoul National University, Seoul, Korea;( z4 I& s t2 y( _/ n! `" q
7 n! ~4 w2 |. hCorrespondence: 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@snu.ac.kr$ Q3 j& x. j: b. R
) ^+ a8 v; u+ G# o" H" W% zABSTRACT 7 b4 q' ^0 S; p3 r! p7 Z. [5 I$ z* J1 \. ~8 f) d, p
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 . 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.; A3 x+ |# ]- I7 ?) b9 Y* R
$ u; }& U- \: t3 r# |' AThe 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 . 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. 7 I+ s' l4 }# N0 W 6 S! ]9 @: l) x; G P/ s9 fMATERIALS AND METHODS 7 e" G M1 a' Z5 J* [, A e . [9 f4 @, ?( O/ jReactivity of PGCs with Single Marker Candidates (Experiments 1 Through 4) m- Y9 H* C# Z/ F( Y+ J) o( g) `3 i, F) M e8 e* V- v- F
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. 1). There were several isolated PGCs or small colonies of PGCs, but those all also reacted with PAS (Figs. 1D, 1F). As shown in Table 1, densitometric quantification also showed a significant difference in the intensity of staining between the PGC colonies and the feeder layers (p 8 r M3 P0 K# j1 y7 v
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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 0 H& k. H U- B: c1 f ! I8 N* @% }% r9 sTable 1. Binding activitya of colony-forming PGCs on days 10 and 40 of culture and mouse ES cells with marker candidates M/ e3 n) d" _& p% l
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Immunostaining demonstrated statistically significant (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.+ p; \# d# {9 C7 u
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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., W- Z$ Q$ i4 z/ r
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In experiment 3, PGCs reacted specifically with anti-integrin 6 and anti-integrin ?1 antibodies (Fig. 3). Densitometric quantification showed that the intensity of staining with both anti-integrin 6 (p - g ^5 _) f) K1 U3 P4 X( l2 }7 M" J: x* O, |$ w
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 6 and (B, D, F, H) anti-integrin ?1 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 6 and ?1 with no non-specific staining of the feeder cell layer. Mouse ES cells were also stained with these antibodies. Scale bar = 50 μm. ( v( u6 U, F. ]& }. R* s+ ~& r: \3 z0 f' ]3 B5 f
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 y: l7 @9 W$ T8 c1 l i3 `3 f1 `% l3 T, H3 w
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.) q0 H; L# R6 j" _& z
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As shown in Figure 5, 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 6 g6 o* d# U, g- Z7 t! e% {; p# @+ l* C
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.! E+ W# a- Y9 N( m5 @
8 ]6 g- x1 e4 G/ A' DDouble Staining with STA and Anti-SSEA Antibodies (Experiment 5) 3 R' L4 D3 S& k! Q. _+ G7 H, W; j6 {9 G0 N/ v! j8 N+ q) D; y. I
The PGCs were stained with anti-SSEA-1, anti-SSEA-3, anti-SSEA-4, anti-integrin 6, or anti-integrin ?1 antibodies in combination with STA. As shown in Figure 6, 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 6, or anti-integrin ?1 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). " l+ d- Y" c8 r+ ^3 s) J. p* @1 z7 m9 d' B. |% ^; w
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 6, or (O) anti-integrin?1 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.+ g+ Q2 c$ o2 ~9 ]
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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. * m, r" f' \" p* V % B9 q: g0 z* tREFERENCES 7 E1 D1 T- f" e; C6 ? D+ p2 l # i. m3 d0 n! s( `0 W( r8 C7 _$ SWentworth BC, Tsai H, Hallett JH et al. Manipulation of avian primordial germ cells and gonadal differentiation. Poult Sci 1989;68:999–1010. 0 X0 S: @) f1 ^0 e- \: J- K; n9 H6 R# O- X
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