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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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Epithelial-like cells expressed TS cell markers.(A) Immunofluorescence staining detected the ubiquitous expression of CDX2, CK7, and CGβ in epithelial-like cells. Note that the expression of CK7 by giant cells was weak (arrow), placental lactogen-1 (PL1) was only detected in giant cells (arrowhead), and Vimentin was absent in all cells. The scale bar represents 100 µm. (B) RT-PCR analysis of the expressions of TS cell markers in epithelial-like cells. Mouse MEF and rabbit ES cell cDNA were used as negative control and cDNA from d20 rabbit placenta was used as positive control. Lane 1, day 20 rabbit placenta sample; Lane 2, rTS cell sample at the 17th passage; Lane 3, rTS cell sample at the 40th passage; Lane 4, mouse embryonic fibroblast sample; Lane 5, rabbit ES cell sample. (C) Western blotting detected the expression of CDX2, CK7, placental lactogen-1 (PL1) and CGβ in rTS-like cells. The GAPDH expression was used as loading control.
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pone-0017124-g002: Epithelial-like cells expressed TS cell markers.(A) Immunofluorescence staining detected the ubiquitous expression of CDX2, CK7, and CGβ in epithelial-like cells. Note that the expression of CK7 by giant cells was weak (arrow), placental lactogen-1 (PL1) was only detected in giant cells (arrowhead), and Vimentin was absent in all cells. The scale bar represents 100 µm. (B) RT-PCR analysis of the expressions of TS cell markers in epithelial-like cells. Mouse MEF and rabbit ES cell cDNA were used as negative control and cDNA from d20 rabbit placenta was used as positive control. Lane 1, day 20 rabbit placenta sample; Lane 2, rTS cell sample at the 17th passage; Lane 3, rTS cell sample at the 40th passage; Lane 4, mouse embryonic fibroblast sample; Lane 5, rabbit ES cell sample. (C) Western blotting detected the expression of CDX2, CK7, placental lactogen-1 (PL1) and CGβ in rTS-like cells. The GAPDH expression was used as loading control.

Mentions: The epithelial-like cells maintained self-renewal during continuous passages, although some cells spontaneously differentiated into giant nuclear cells (Figure 1D, arrow). These properties prompted us to examine if these cells are trophectodermal lineage stem cells. The mRNA expressions of Oct4, Nanog, Sox2 (three pluripotency genes), Cdx2, Esrrb, Eomes (three transcription factors characteristic of the trophectoderm in mouse and vole), Hand1 (known to promote the differentiation of giant trophoblast cells and is highly expressed in undifferentiated and differentiated mouse and vole TS cells), Gcm1 (syncytiotrophoblast marker) and Tpbpa (specific for spongiotrophoblast and ectoplacental cone) were examined by RT-PCR [7], [16], [17], [18]. As shown (Figure S1), the epithelial-like cells highly expressed the pluripotency marker Oct4 but not Nanog or Sox2 at passage 17. However, the expression level of Oct4 decreased dramatically at passage 40, as that of Gcm1 (Figure 2B), which might suggest stress [19]. The trophoblastic lineage markers Cdx2, Eomes, Hand1 and Gcm1 were consistently detected in epithelial-like cells at passages 17 (P17) and 40 (P40). Tpbpa mRNAs were expressed in rabbit placenta but not in epithelial-like cells. Surprisingly, the transcription factor Esrrb, which is expressed in TS cells and placenta of both mouse and vole [17], was not detected in either rabbit placenta or epithelia-like cells. Further studies were needed to verify the expression of Esrrb in rabbit. In consistency with the mRNA expression data, immunofluorescent staining and western blotting detected the expression of cytokeratin-7 (epithelial marker), CDX2, and chorionic gonadotropin β subunit (CG-β, trophoblast marker) [10] in these cells (Figure 2A, 2C). The germ layer markers Vimentin (Figure 2A), Nestin or Brachyury were not detected in these cells (data not shown). In accordance to the observation of spontaneous differentiation, Placental lactogen-I (PL-I), a specific marker for giant cells [20], was occasionally detected in some cells after prolonged culture by immunofluorescent staining (Figure 2A). Taken together, these data suggested that these epithelial-like cells were TS-like cells.


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)

Epithelial-like cells expressed TS cell markers.(A) Immunofluorescence staining detected the ubiquitous expression of CDX2, CK7, and CGβ in epithelial-like cells. Note that the expression of CK7 by giant cells was weak (arrow), placental lactogen-1 (PL1) was only detected in giant cells (arrowhead), and Vimentin was absent in all cells. The scale bar represents 100 µm. (B) RT-PCR analysis of the expressions of TS cell markers in epithelial-like cells. Mouse MEF and rabbit ES cell cDNA were used as negative control and cDNA from d20 rabbit placenta was used as positive control. Lane 1, day 20 rabbit placenta sample; Lane 2, rTS cell sample at the 17th passage; Lane 3, rTS cell sample at the 40th passage; Lane 4, mouse embryonic fibroblast sample; Lane 5, rabbit ES cell sample. (C) Western blotting detected the expression of CDX2, CK7, placental lactogen-1 (PL1) and CGβ in rTS-like cells. The GAPDH expression was used as loading control.
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pone-0017124-g002: Epithelial-like cells expressed TS cell markers.(A) Immunofluorescence staining detected the ubiquitous expression of CDX2, CK7, and CGβ in epithelial-like cells. Note that the expression of CK7 by giant cells was weak (arrow), placental lactogen-1 (PL1) was only detected in giant cells (arrowhead), and Vimentin was absent in all cells. The scale bar represents 100 µm. (B) RT-PCR analysis of the expressions of TS cell markers in epithelial-like cells. Mouse MEF and rabbit ES cell cDNA were used as negative control and cDNA from d20 rabbit placenta was used as positive control. Lane 1, day 20 rabbit placenta sample; Lane 2, rTS cell sample at the 17th passage; Lane 3, rTS cell sample at the 40th passage; Lane 4, mouse embryonic fibroblast sample; Lane 5, rabbit ES cell sample. (C) Western blotting detected the expression of CDX2, CK7, placental lactogen-1 (PL1) and CGβ in rTS-like cells. The GAPDH expression was used as loading control.
Mentions: The epithelial-like cells maintained self-renewal during continuous passages, although some cells spontaneously differentiated into giant nuclear cells (Figure 1D, arrow). These properties prompted us to examine if these cells are trophectodermal lineage stem cells. The mRNA expressions of Oct4, Nanog, Sox2 (three pluripotency genes), Cdx2, Esrrb, Eomes (three transcription factors characteristic of the trophectoderm in mouse and vole), Hand1 (known to promote the differentiation of giant trophoblast cells and is highly expressed in undifferentiated and differentiated mouse and vole TS cells), Gcm1 (syncytiotrophoblast marker) and Tpbpa (specific for spongiotrophoblast and ectoplacental cone) were examined by RT-PCR [7], [16], [17], [18]. As shown (Figure S1), the epithelial-like cells highly expressed the pluripotency marker Oct4 but not Nanog or Sox2 at passage 17. However, the expression level of Oct4 decreased dramatically at passage 40, as that of Gcm1 (Figure 2B), which might suggest stress [19]. The trophoblastic lineage markers Cdx2, Eomes, Hand1 and Gcm1 were consistently detected in epithelial-like cells at passages 17 (P17) and 40 (P40). Tpbpa mRNAs were expressed in rabbit placenta but not in epithelial-like cells. Surprisingly, the transcription factor Esrrb, which is expressed in TS cells and placenta of both mouse and vole [17], was not detected in either rabbit placenta or epithelia-like cells. Further studies were needed to verify the expression of Esrrb in rabbit. In consistency with the mRNA expression data, immunofluorescent staining and western blotting detected the expression of cytokeratin-7 (epithelial marker), CDX2, and chorionic gonadotropin β subunit (CG-β, trophoblast marker) [10] in these cells (Figure 2A, 2C). The germ layer markers Vimentin (Figure 2A), Nestin or Brachyury were not detected in these cells (data not shown). In accordance to the observation of spontaneous differentiation, Placental lactogen-I (PL-I), a specific marker for giant cells [20], was occasionally detected in some cells after prolonged culture by immunofluorescent staining (Figure 2A). Taken together, these data suggested that these epithelial-like cells were TS-like cells.

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