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Primordial germ cell specification from embryonic stem cells.

Wei W, Qing T, Ye X, Liu H, Zhang D, Yang W, Deng H - PLoS ONE (2008)

Bottom Line: However, neither Stella-positive ES cells nor Stella-negative ES cells shared a similar gene expression pattern with either PGCs or PGC precursors.Compared with PGCs derived via the attachment culture technique, PGCs derived via the EB method that had undergone the sequential erasure of Peg3 followed by Igf2r resulted in a cell line in which the expression dynamics of T, Fgf8 and Sox17, in addition to the expression of the epiblast markers, were more similar to the in vivo expression, thus demonstrating that the process of PGC derivation was more faithfully recapitulated using the EB method.Furthermore, we developed an in vitro model of PGC specification in a completely chemically defined medium (CDM) that indicated that BMP4 and Wnt3a promoted PGC derivation, whereas BMP8b and activinA had no observable effect on PGC derivation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Chemical Genomics, School of Chemical Biology and Biothechnology, Shenzhen Graduate School of Peking University, Shenzhen, China.

ABSTRACT

Background: Primordial germ cell (PGC) specification is the first crucial step in germ line development. However, owing to significant challenges regarding the in vivo system, such as the complex cellular environment and potential problems with embryo manipulation, it is desirable to generate embryonic stem (ES) cells that are capable of overcoming these aforementioned limitations in order to provide a potential in vitro model to recapitulate the developmental processes in vivo.

Methodology and principal findings: Here, we studied the detailed process of PGC specification from stella-GFP ES cells. We first observed the heterogeneous expression of stella in ES cells. However, neither Stella-positive ES cells nor Stella-negative ES cells shared a similar gene expression pattern with either PGCs or PGC precursors. Second, we derived PGCs from ES cells using two differentiation methods, namely the attachment culture technique and the embryoid body (EB) method. Compared with PGCs derived via the attachment culture technique, PGCs derived via the EB method that had undergone the sequential erasure of Peg3 followed by Igf2r resulted in a cell line in which the expression dynamics of T, Fgf8 and Sox17, in addition to the expression of the epiblast markers, were more similar to the in vivo expression, thus demonstrating that the process of PGC derivation was more faithfully recapitulated using the EB method. Furthermore, we developed an in vitro model of PGC specification in a completely chemically defined medium (CDM) that indicated that BMP4 and Wnt3a promoted PGC derivation, whereas BMP8b and activinA had no observable effect on PGC derivation.

Conclusions and significance: The in vitro model we have established can recapitulate the developmental processes in vivo and provides new insights into the mechanism of PGC specification.

Show MeSH
Induction of PGCs by BMP4 in the CDM.(A) Flow cytometric analysis of stella-GFP expression in day 4 EBs in the CDM or supplemented with BMP4, noggin or BMP4+ noggin. (B) Gene-expression analysis of PGC markers in GFP positive cells in (A). The relative expression of each gene in differentiated cultures was normalized by its expression in GFP-positive cells in the CDM with BMP4 after normalization with Gapdh. (C). Gene-expression analysis in GFP-positive (BMP4+) and GFP-negative (BMP4−) cells in day 4 EBs in the CDM with BMP4. Foxa2 is a mesoendoderm marker, whereas Mixl1, Flk1, Mesp1, Evx1 and Tbx6 are mesoderm markers [24], [25]. Gapdh served as loading control.
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pone-0004013-g004: Induction of PGCs by BMP4 in the CDM.(A) Flow cytometric analysis of stella-GFP expression in day 4 EBs in the CDM or supplemented with BMP4, noggin or BMP4+ noggin. (B) Gene-expression analysis of PGC markers in GFP positive cells in (A). The relative expression of each gene in differentiated cultures was normalized by its expression in GFP-positive cells in the CDM with BMP4 after normalization with Gapdh. (C). Gene-expression analysis in GFP-positive (BMP4+) and GFP-negative (BMP4−) cells in day 4 EBs in the CDM with BMP4. Foxa2 is a mesoendoderm marker, whereas Mixl1, Flk1, Mesp1, Evx1 and Tbx6 are mesoderm markers [24], [25]. Gapdh served as loading control.

Mentions: To determine the signals that promote the derivation of PGCs, we attempted to develop an in vitro model of PGC specification. First, because the two subpopulations of stella-GFP ES cells were interchangeable regarding stella expression (Figs. 1B and C), we did not sort either of them to establish the model. Second, because the process of PGC derivation was more faithfully recapitulated in the EB method than in the attachment culture technique, we employed the EB method. Third, because unknown components coupled with the inherent variability in the quality of the serum are known to hamper the accuracy of the results [15], we decided to develop a completely chemically defined medium (CDM). In this respect, several basic media (including DMEM, DMEM/F12, Ham's F12, X-vivo, 1640, and IMDM) were tested, and we found that a combination of Ham's F12 with IMDM supported the survival of cells most effectively. Because the relative percentage of GFP-positive cells was extremely low in EBs formed in the CDM (0.78%) (Fig. 4A), the CDM model provided a strategy to study the signals that trigger PGC derivation.


Primordial germ cell specification from embryonic stem cells.

Wei W, Qing T, Ye X, Liu H, Zhang D, Yang W, Deng H - PLoS ONE (2008)

Induction of PGCs by BMP4 in the CDM.(A) Flow cytometric analysis of stella-GFP expression in day 4 EBs in the CDM or supplemented with BMP4, noggin or BMP4+ noggin. (B) Gene-expression analysis of PGC markers in GFP positive cells in (A). The relative expression of each gene in differentiated cultures was normalized by its expression in GFP-positive cells in the CDM with BMP4 after normalization with Gapdh. (C). Gene-expression analysis in GFP-positive (BMP4+) and GFP-negative (BMP4−) cells in day 4 EBs in the CDM with BMP4. Foxa2 is a mesoendoderm marker, whereas Mixl1, Flk1, Mesp1, Evx1 and Tbx6 are mesoderm markers [24], [25]. Gapdh served as loading control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2602984&req=5

pone-0004013-g004: Induction of PGCs by BMP4 in the CDM.(A) Flow cytometric analysis of stella-GFP expression in day 4 EBs in the CDM or supplemented with BMP4, noggin or BMP4+ noggin. (B) Gene-expression analysis of PGC markers in GFP positive cells in (A). The relative expression of each gene in differentiated cultures was normalized by its expression in GFP-positive cells in the CDM with BMP4 after normalization with Gapdh. (C). Gene-expression analysis in GFP-positive (BMP4+) and GFP-negative (BMP4−) cells in day 4 EBs in the CDM with BMP4. Foxa2 is a mesoendoderm marker, whereas Mixl1, Flk1, Mesp1, Evx1 and Tbx6 are mesoderm markers [24], [25]. Gapdh served as loading control.
Mentions: To determine the signals that promote the derivation of PGCs, we attempted to develop an in vitro model of PGC specification. First, because the two subpopulations of stella-GFP ES cells were interchangeable regarding stella expression (Figs. 1B and C), we did not sort either of them to establish the model. Second, because the process of PGC derivation was more faithfully recapitulated in the EB method than in the attachment culture technique, we employed the EB method. Third, because unknown components coupled with the inherent variability in the quality of the serum are known to hamper the accuracy of the results [15], we decided to develop a completely chemically defined medium (CDM). In this respect, several basic media (including DMEM, DMEM/F12, Ham's F12, X-vivo, 1640, and IMDM) were tested, and we found that a combination of Ham's F12 with IMDM supported the survival of cells most effectively. Because the relative percentage of GFP-positive cells was extremely low in EBs formed in the CDM (0.78%) (Fig. 4A), the CDM model provided a strategy to study the signals that trigger PGC derivation.

Bottom Line: However, neither Stella-positive ES cells nor Stella-negative ES cells shared a similar gene expression pattern with either PGCs or PGC precursors.Compared with PGCs derived via the attachment culture technique, PGCs derived via the EB method that had undergone the sequential erasure of Peg3 followed by Igf2r resulted in a cell line in which the expression dynamics of T, Fgf8 and Sox17, in addition to the expression of the epiblast markers, were more similar to the in vivo expression, thus demonstrating that the process of PGC derivation was more faithfully recapitulated using the EB method.Furthermore, we developed an in vitro model of PGC specification in a completely chemically defined medium (CDM) that indicated that BMP4 and Wnt3a promoted PGC derivation, whereas BMP8b and activinA had no observable effect on PGC derivation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Chemical Genomics, School of Chemical Biology and Biothechnology, Shenzhen Graduate School of Peking University, Shenzhen, China.

ABSTRACT

Background: Primordial germ cell (PGC) specification is the first crucial step in germ line development. However, owing to significant challenges regarding the in vivo system, such as the complex cellular environment and potential problems with embryo manipulation, it is desirable to generate embryonic stem (ES) cells that are capable of overcoming these aforementioned limitations in order to provide a potential in vitro model to recapitulate the developmental processes in vivo.

Methodology and principal findings: Here, we studied the detailed process of PGC specification from stella-GFP ES cells. We first observed the heterogeneous expression of stella in ES cells. However, neither Stella-positive ES cells nor Stella-negative ES cells shared a similar gene expression pattern with either PGCs or PGC precursors. Second, we derived PGCs from ES cells using two differentiation methods, namely the attachment culture technique and the embryoid body (EB) method. Compared with PGCs derived via the attachment culture technique, PGCs derived via the EB method that had undergone the sequential erasure of Peg3 followed by Igf2r resulted in a cell line in which the expression dynamics of T, Fgf8 and Sox17, in addition to the expression of the epiblast markers, were more similar to the in vivo expression, thus demonstrating that the process of PGC derivation was more faithfully recapitulated using the EB method. Furthermore, we developed an in vitro model of PGC specification in a completely chemically defined medium (CDM) that indicated that BMP4 and Wnt3a promoted PGC derivation, whereas BMP8b and activinA had no observable effect on PGC derivation.

Conclusions and significance: The in vitro model we have established can recapitulate the developmental processes in vivo and provides new insights into the mechanism of PGC specification.

Show MeSH