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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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Rabbit TS-like cells can differentiate into trophoblastic derivatives in vitro.(A) Semi-quantitative RT-PCR analysis detected the increase in mRNA expression of Gcm1 (marker of syncytiotrophoblast) and the decrease of Cdx2 expression (trophoblast stem cell marker) in TS-like cells treated with different concentrations of dbcAMP. (B) Western blotting detected the expression of relaxin (syncytiotrophoblast marker) inTS-like cells treated with different concentrations of dbcAMP. (C) The TS-like cells cultured on Matrigel coated transwell invaded the transwell membrane. Fluorescent image (Hoechst 33342) and merged image of Hoechst 33342 with differential interference contrast (Dic) is shown. Arrows showed the giant nuclei of the penetrated cells and arrowheads showed the small nuclei of the cells. Red arrow indicated the pore of the transwell membrane. All analyses were repeated three times, and the representative figures are shown here.
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pone-0017124-g003: Rabbit TS-like cells can differentiate into trophoblastic derivatives in vitro.(A) Semi-quantitative RT-PCR analysis detected the increase in mRNA expression of Gcm1 (marker of syncytiotrophoblast) and the decrease of Cdx2 expression (trophoblast stem cell marker) in TS-like cells treated with different concentrations of dbcAMP. (B) Western blotting detected the expression of relaxin (syncytiotrophoblast marker) inTS-like cells treated with different concentrations of dbcAMP. (C) The TS-like cells cultured on Matrigel coated transwell invaded the transwell membrane. Fluorescent image (Hoechst 33342) and merged image of Hoechst 33342 with differential interference contrast (Dic) is shown. Arrows showed the giant nuclei of the penetrated cells and arrowheads showed the small nuclei of the cells. Red arrow indicated the pore of the transwell membrane. All analyses were repeated three times, and the representative figures are shown here.

Mentions: To further clarify the identity of these TS-like cells, we went on to investigate if they have TS cell abilities to differentiate into trophoblast subtypes in vitro, and to chimerize placental tissues in vivo [21], [22], [23]. TS-like cells were treated with dibutyryl cAMP (dbcAMP) (0 mM, 2 mM and 4 mM) to induce differentiation into syncytiotrophoblast [24]. dbcAMP promoted transformation of TS-like cells into multinucleated cells in a concentration-independent manner. Similarly, time lapse microscopy revealed that adherent TS-like cells occasionally formed multinucleated syncytiotrophoblast when they met each other. Syncytiotrophoblasts were also formed through the fusion of mononucleated daughter cells (Movie S1). In accordance with the morphological change, the expression of the genes specific to the differentiated syncytiotrophoblast increased under the drug treatment. As shown, addition of dbcAMP increased Gcm1 expression and decreased CDX2 expression as detected by semi quantitative RT-PCR (Figure 3A). The protein level of relaxin, a marker of rabbit placental syncytiotrophoblast cells [24], was also elevated by dbcAMP treatment (Figure 3B). Moreover, the secretion of hormones (chorionic gonadotropin, progesterone and estradiol) by TS-like cells could be detected in the culture medium (Figure S2).


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)

Rabbit TS-like cells can differentiate into trophoblastic derivatives in vitro.(A) Semi-quantitative RT-PCR analysis detected the increase in mRNA expression of Gcm1 (marker of syncytiotrophoblast) and the decrease of Cdx2 expression (trophoblast stem cell marker) in TS-like cells treated with different concentrations of dbcAMP. (B) Western blotting detected the expression of relaxin (syncytiotrophoblast marker) inTS-like cells treated with different concentrations of dbcAMP. (C) The TS-like cells cultured on Matrigel coated transwell invaded the transwell membrane. Fluorescent image (Hoechst 33342) and merged image of Hoechst 33342 with differential interference contrast (Dic) is shown. Arrows showed the giant nuclei of the penetrated cells and arrowheads showed the small nuclei of the cells. Red arrow indicated the pore of the transwell membrane. All analyses were repeated three times, and the representative figures are shown here.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017124-g003: Rabbit TS-like cells can differentiate into trophoblastic derivatives in vitro.(A) Semi-quantitative RT-PCR analysis detected the increase in mRNA expression of Gcm1 (marker of syncytiotrophoblast) and the decrease of Cdx2 expression (trophoblast stem cell marker) in TS-like cells treated with different concentrations of dbcAMP. (B) Western blotting detected the expression of relaxin (syncytiotrophoblast marker) inTS-like cells treated with different concentrations of dbcAMP. (C) The TS-like cells cultured on Matrigel coated transwell invaded the transwell membrane. Fluorescent image (Hoechst 33342) and merged image of Hoechst 33342 with differential interference contrast (Dic) is shown. Arrows showed the giant nuclei of the penetrated cells and arrowheads showed the small nuclei of the cells. Red arrow indicated the pore of the transwell membrane. All analyses were repeated three times, and the representative figures are shown here.
Mentions: To further clarify the identity of these TS-like cells, we went on to investigate if they have TS cell abilities to differentiate into trophoblast subtypes in vitro, and to chimerize placental tissues in vivo [21], [22], [23]. TS-like cells were treated with dibutyryl cAMP (dbcAMP) (0 mM, 2 mM and 4 mM) to induce differentiation into syncytiotrophoblast [24]. dbcAMP promoted transformation of TS-like cells into multinucleated cells in a concentration-independent manner. Similarly, time lapse microscopy revealed that adherent TS-like cells occasionally formed multinucleated syncytiotrophoblast when they met each other. Syncytiotrophoblasts were also formed through the fusion of mononucleated daughter cells (Movie S1). In accordance with the morphological change, the expression of the genes specific to the differentiated syncytiotrophoblast increased under the drug treatment. As shown, addition of dbcAMP increased Gcm1 expression and decreased CDX2 expression as detected by semi quantitative RT-PCR (Figure 3A). The protein level of relaxin, a marker of rabbit placental syncytiotrophoblast cells [24], was also elevated by dbcAMP treatment (Figure 3B). Moreover, the secretion of hormones (chorionic gonadotropin, progesterone and estradiol) by TS-like cells could be detected in the culture medium (Figure S2).

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