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Generation of trophoblast stem cells from rabbit embryonic stem cells with BMP4.

Tan T, Tang X, Zhang J, Niu Y, Chen H, Li B, Wei Q, Ji W - PLoS ONE (2011)

Bottom Line: Trophoblast stem (TS) cells are ideal models to investigate trophectoderm differentiation and placental development.Rabbit ES cells generated in our laboratory were induced to differentiate in the presence of BMP4 and TS-like cell colonies were isolated and expanded.These cells expressed the molecular markers of mouse TS cells, were able to invade, give rise to derivatives of TS cells, and chimerize placental tissues when injected into blastocysts.

View Article: PubMed Central - PubMed

Affiliation: Kunming Primate Research Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China.

ABSTRACT
Trophoblast stem (TS) cells are ideal models to investigate trophectoderm differentiation and placental development. Herein, we describe the derivation of rabbit trophoblast stem cells from embryonic stem (ES) cells. Rabbit ES cells generated in our laboratory were induced to differentiate in the presence of BMP4 and TS-like cell colonies were isolated and expanded. These cells expressed the molecular markers of mouse TS cells, were able to invade, give rise to derivatives of TS cells, and chimerize placental tissues when injected into blastocysts. The rabbit TS-like cells maintained self-renewal in culture medium with serum but without growth factors or feeder cells, whilst their proliferation and identity were compromised by inhibitors of FGFs and TGF-β receptors. Taken together, our study demonstrated the derivation of rabbit TS cells and suggested the essential roles of FGF and TGF-β signalings in maintenance of rabbit TS cell self-renewal.

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FGF and TGF-β signalings are essential for rabbit TS-like cells self-renewal.(A) RT-PCR detected the mRNA expressions of the key components of FGF and TGF-β signalings in TS-like cells. Lane 1, sample of rabbit ES cells; Lane 2, sample of TS-like cells at 17th passage; Lane 3, sample of TS-like cells at 40th passage. (B) FACS examination of cell cycle stages (G1, G2, and S phases) after TS-like cells were treated with growth factors for 48 hours in the presence or absence of serum. Note that in the absence of serum, growth factors stimulate cell proliferation (n = 3, * represents P<0.05). (C) FACS examination of cell cycle stages (G1, G2, and S phases) after TS-like cells were treated with growth factor inhibitors (20 µM of SB431542 against TGF-β type I receptor or 20 µM SU5402 against FGF receptor 1) for 48 hours in the serum-free medium (SF). Note that growth factor inhibitor treatments increased the percentages of TS-like cells at G1 phase (n = 3, * represents P<0.05). (D) Semi-quantitative RT-PCR analysis of mRNA expressions of the genes specific to trophoblastic lineage in TS-like cells cultured in ES medium (ES), ES medium with TGF-β (ES+TGF-β), serum free medium (SF), serum free medium with SB431542 (SF+SB), or serum free medium with SU5402 (SF+SU).
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pone-0017124-g005: FGF and TGF-β signalings are essential for rabbit TS-like cells self-renewal.(A) RT-PCR detected the mRNA expressions of the key components of FGF and TGF-β signalings in TS-like cells. Lane 1, sample of rabbit ES cells; Lane 2, sample of TS-like cells at 17th passage; Lane 3, sample of TS-like cells at 40th passage. (B) FACS examination of cell cycle stages (G1, G2, and S phases) after TS-like cells were treated with growth factors for 48 hours in the presence or absence of serum. Note that in the absence of serum, growth factors stimulate cell proliferation (n = 3, * represents P<0.05). (C) FACS examination of cell cycle stages (G1, G2, and S phases) after TS-like cells were treated with growth factor inhibitors (20 µM of SB431542 against TGF-β type I receptor or 20 µM SU5402 against FGF receptor 1) for 48 hours in the serum-free medium (SF). Note that growth factor inhibitor treatments increased the percentages of TS-like cells at G1 phase (n = 3, * represents P<0.05). (D) Semi-quantitative RT-PCR analysis of mRNA expressions of the genes specific to trophoblastic lineage in TS-like cells cultured in ES medium (ES), ES medium with TGF-β (ES+TGF-β), serum free medium (SF), serum free medium with SB431542 (SF+SB), or serum free medium with SU5402 (SF+SU).

Mentions: Previous studies reported the essential roles of FGF and TGF-β signaling in maintenance of mouse TS cell self-renewal [16], [26]. In this study, rabbit TS-like cells were propagated in culture medium supplemented with FBS but without growth factors under the feeder-cell free condition. To clarify if TS-like cell proliferation was regulated by FGF and/or TGFβ signaling, we cultured TS-like cells in the presence or absence of FBS supplied with or without these growth factors. Cell proliferation was monitored by the change of DNA content. As shown, the proliferation rate of TS-like cells was attenuated after withdrawal of FBS. The attenuation could be partially reversed by addition of 2 ng/ml TGF-β1 or 25 ng/ml aFGF+25 ng/ml bFGF into serum-free medium. However, addition of growth factors in the presence of FBS did not affect the proliferation rate (Figure 5B). These data suggested that FGF and TGF-β signalings were beneficial to TS-like cell proliferation, and their activation could be triggered by serum. To further confirm the indispensable roles of FGF and TGF-β signalings in the maintenance of TS-like cells, we treated the cells with FGF receptor 1 (FGFR1) inhibitor SU5402 or TGF-β type I receptor inhibitor SB431542 [27] to interrupt the signaling. In the absence of FBS, interfering either of the signaling pathways increased the population of cells at the G1 phase (Figure 5C), indicating that the proliferation of the TS-like cells was dependent of the two signalings. Concordantly, in TS-like cells we detected the mRNA expression of some components of FGF pathway (FGFR2, FGFR3, FGFR4, SOS1, PTPN11) and TGF-β pathway (Smad1, 2, 3, 4) (Figure 5A).


Generation of trophoblast stem cells from rabbit embryonic stem cells with BMP4.

Tan T, Tang X, Zhang J, Niu Y, Chen H, Li B, Wei Q, Ji W - PLoS ONE (2011)

FGF and TGF-β signalings are essential for rabbit TS-like cells self-renewal.(A) RT-PCR detected the mRNA expressions of the key components of FGF and TGF-β signalings in TS-like cells. Lane 1, sample of rabbit ES cells; Lane 2, sample of TS-like cells at 17th passage; Lane 3, sample of TS-like cells at 40th passage. (B) FACS examination of cell cycle stages (G1, G2, and S phases) after TS-like cells were treated with growth factors for 48 hours in the presence or absence of serum. Note that in the absence of serum, growth factors stimulate cell proliferation (n = 3, * represents P<0.05). (C) FACS examination of cell cycle stages (G1, G2, and S phases) after TS-like cells were treated with growth factor inhibitors (20 µM of SB431542 against TGF-β type I receptor or 20 µM SU5402 against FGF receptor 1) for 48 hours in the serum-free medium (SF). Note that growth factor inhibitor treatments increased the percentages of TS-like cells at G1 phase (n = 3, * represents P<0.05). (D) Semi-quantitative RT-PCR analysis of mRNA expressions of the genes specific to trophoblastic lineage in TS-like cells cultured in ES medium (ES), ES medium with TGF-β (ES+TGF-β), serum free medium (SF), serum free medium with SB431542 (SF+SB), or serum free medium with SU5402 (SF+SU).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040765&req=5

pone-0017124-g005: FGF and TGF-β signalings are essential for rabbit TS-like cells self-renewal.(A) RT-PCR detected the mRNA expressions of the key components of FGF and TGF-β signalings in TS-like cells. Lane 1, sample of rabbit ES cells; Lane 2, sample of TS-like cells at 17th passage; Lane 3, sample of TS-like cells at 40th passage. (B) FACS examination of cell cycle stages (G1, G2, and S phases) after TS-like cells were treated with growth factors for 48 hours in the presence or absence of serum. Note that in the absence of serum, growth factors stimulate cell proliferation (n = 3, * represents P<0.05). (C) FACS examination of cell cycle stages (G1, G2, and S phases) after TS-like cells were treated with growth factor inhibitors (20 µM of SB431542 against TGF-β type I receptor or 20 µM SU5402 against FGF receptor 1) for 48 hours in the serum-free medium (SF). Note that growth factor inhibitor treatments increased the percentages of TS-like cells at G1 phase (n = 3, * represents P<0.05). (D) Semi-quantitative RT-PCR analysis of mRNA expressions of the genes specific to trophoblastic lineage in TS-like cells cultured in ES medium (ES), ES medium with TGF-β (ES+TGF-β), serum free medium (SF), serum free medium with SB431542 (SF+SB), or serum free medium with SU5402 (SF+SU).
Mentions: Previous studies reported the essential roles of FGF and TGF-β signaling in maintenance of mouse TS cell self-renewal [16], [26]. In this study, rabbit TS-like cells were propagated in culture medium supplemented with FBS but without growth factors under the feeder-cell free condition. To clarify if TS-like cell proliferation was regulated by FGF and/or TGFβ signaling, we cultured TS-like cells in the presence or absence of FBS supplied with or without these growth factors. Cell proliferation was monitored by the change of DNA content. As shown, the proliferation rate of TS-like cells was attenuated after withdrawal of FBS. The attenuation could be partially reversed by addition of 2 ng/ml TGF-β1 or 25 ng/ml aFGF+25 ng/ml bFGF into serum-free medium. However, addition of growth factors in the presence of FBS did not affect the proliferation rate (Figure 5B). These data suggested that FGF and TGF-β signalings were beneficial to TS-like cell proliferation, and their activation could be triggered by serum. To further confirm the indispensable roles of FGF and TGF-β signalings in the maintenance of TS-like cells, we treated the cells with FGF receptor 1 (FGFR1) inhibitor SU5402 or TGF-β type I receptor inhibitor SB431542 [27] to interrupt the signaling. In the absence of FBS, interfering either of the signaling pathways increased the population of cells at the G1 phase (Figure 5C), indicating that the proliferation of the TS-like cells was dependent of the two signalings. Concordantly, in TS-like cells we detected the mRNA expression of some components of FGF pathway (FGFR2, FGFR3, FGFR4, SOS1, PTPN11) and TGF-β pathway (Smad1, 2, 3, 4) (Figure 5A).

Bottom Line: Trophoblast stem (TS) cells are ideal models to investigate trophectoderm differentiation and placental development.Rabbit ES cells generated in our laboratory were induced to differentiate in the presence of BMP4 and TS-like cell colonies were isolated and expanded.These cells expressed the molecular markers of mouse TS cells, were able to invade, give rise to derivatives of TS cells, and chimerize placental tissues when injected into blastocysts.

View Article: PubMed Central - PubMed

Affiliation: Kunming Primate Research Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China.

ABSTRACT
Trophoblast stem (TS) cells are ideal models to investigate trophectoderm differentiation and placental development. Herein, we describe the derivation of rabbit trophoblast stem cells from embryonic stem (ES) cells. Rabbit ES cells generated in our laboratory were induced to differentiate in the presence of BMP4 and TS-like cell colonies were isolated and expanded. These cells expressed the molecular markers of mouse TS cells, were able to invade, give rise to derivatives of TS cells, and chimerize placental tissues when injected into blastocysts. The rabbit TS-like cells maintained self-renewal in culture medium with serum but without growth factors or feeder cells, whilst their proliferation and identity were compromised by inhibitors of FGFs and TGF-β receptors. Taken together, our study demonstrated the derivation of rabbit TS cells and suggested the essential roles of FGF and TGF-β signalings in maintenance of rabbit TS cell self-renewal.

Show MeSH
Related in: MedlinePlus