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Long-term expandable SOX9+ chondrogenic ectomesenchymal cells from human pluripotent stem cells.

Umeda K, Oda H, Yan Q, Matthias N, Zhao J, Davis BR, Nakayama N - Stem Cell Reports (2015)

Bottom Line: When "primed" with TGFβ, such cells efficiently formed translucent cartilage particles, which were completely mineralized in 12 weeks in immunocompromized mice.Ectomesenchyme is a source of many craniofacial bone and cartilage structures.The method we describe for obtaining a large quantity of human ectomesenchymal cells will help to model craniofacial disorders in vitro and potentially provide cells for the repair of craniofacial damage.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine, The University of Texas Health Science Center at Houston Medical School, Houston, TX 77030, USA.

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Pretreatment with TGFβ Facilitates Cartilage Particle Formation from the FSB-Expanded Ectomesenchymal Cells(A and B) Cartilage particle formation from the H9 hESC-derived ectomesenchymal cells maintained under F (A) or FSB (B) by 3D-pellet culture. Toluidine Blue staining (purple) of sections of the cartilage particles, except for (Bvi), which is COL2 immunostained (brown). Some cultures were passaged once or twice (p2) to FT (CDM plus FGF2+TGFβ3) (Aiii, Bi, Bii, Bv, Bvi), or D10 (Aiv, Biii, Biv), prior to pellet culture.(C) Capacity of sGAG production in cartilage particles. The ectomesenchymal cells cultured as indicated (→FT, passage to FT) were subjected to pellet culture and total DNA and sGAGs were quantified. Negative control: undifferentiated ESCs. ∗p < 0.05 (n = 3 independent cultures, one pellet/culture mean ± SD).(D) Upregulation of N-cadherin surface expression on the FSB-expanded ectomesenchymal cells during FT treatment. The ectomesenchymal cells maintained under F (Fp2) and FSB (FSBp5) were either passaged to FSB (→FSB), FT (→FT, FTp2), or D10 (→D10) or not passaged (Fp2), and then FACS analyzed.(E) Effect of FT treatment on SOX9 protein expression. FSB-expanded p7 ectomesenchymal cells followed by FT culture (i.e., p8) were treated and analyzed as in Figure 3D. Graph, n = 4 technical repeats, mean ± SD. Isotype control, Figure S4J.(F) Real-time RT-PCR analysis for investigating the effect of FT treatment on SOX9 gene expression (n = 3 technical repeats, mean ± SD).
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fig4: Pretreatment with TGFβ Facilitates Cartilage Particle Formation from the FSB-Expanded Ectomesenchymal Cells(A and B) Cartilage particle formation from the H9 hESC-derived ectomesenchymal cells maintained under F (A) or FSB (B) by 3D-pellet culture. Toluidine Blue staining (purple) of sections of the cartilage particles, except for (Bvi), which is COL2 immunostained (brown). Some cultures were passaged once or twice (p2) to FT (CDM plus FGF2+TGFβ3) (Aiii, Bi, Bii, Bv, Bvi), or D10 (Aiv, Biii, Biv), prior to pellet culture.(C) Capacity of sGAG production in cartilage particles. The ectomesenchymal cells cultured as indicated (→FT, passage to FT) were subjected to pellet culture and total DNA and sGAGs were quantified. Negative control: undifferentiated ESCs. ∗p < 0.05 (n = 3 independent cultures, one pellet/culture mean ± SD).(D) Upregulation of N-cadherin surface expression on the FSB-expanded ectomesenchymal cells during FT treatment. The ectomesenchymal cells maintained under F (Fp2) and FSB (FSBp5) were either passaged to FSB (→FSB), FT (→FT, FTp2), or D10 (→D10) or not passaged (Fp2), and then FACS analyzed.(E) Effect of FT treatment on SOX9 protein expression. FSB-expanded p7 ectomesenchymal cells followed by FT culture (i.e., p8) were treated and analyzed as in Figure 3D. Graph, n = 4 technical repeats, mean ± SD. Isotype control, Figure S4J.(F) Real-time RT-PCR analysis for investigating the effect of FT treatment on SOX9 gene expression (n = 3 technical repeats, mean ± SD).

Mentions: The ectomesenchymal cells expanded in CDM in the presence of FGF2 or FGF2+SB431542 were subjected to 3D-pellet culture under the PDGF/TGFβ/BMP condition. The early (p2) FGF2-expanded cells formed a large cartilage particle that stained metachromatically (pink to purple) with Toluidine Blue, but then lost such chondrogenic activity by p4–p5 (Figures 4A and 4C), in keeping with the loss of the CD271+ cell population (Figures 2C and S3D).


Long-term expandable SOX9+ chondrogenic ectomesenchymal cells from human pluripotent stem cells.

Umeda K, Oda H, Yan Q, Matthias N, Zhao J, Davis BR, Nakayama N - Stem Cell Reports (2015)

Pretreatment with TGFβ Facilitates Cartilage Particle Formation from the FSB-Expanded Ectomesenchymal Cells(A and B) Cartilage particle formation from the H9 hESC-derived ectomesenchymal cells maintained under F (A) or FSB (B) by 3D-pellet culture. Toluidine Blue staining (purple) of sections of the cartilage particles, except for (Bvi), which is COL2 immunostained (brown). Some cultures were passaged once or twice (p2) to FT (CDM plus FGF2+TGFβ3) (Aiii, Bi, Bii, Bv, Bvi), or D10 (Aiv, Biii, Biv), prior to pellet culture.(C) Capacity of sGAG production in cartilage particles. The ectomesenchymal cells cultured as indicated (→FT, passage to FT) were subjected to pellet culture and total DNA and sGAGs were quantified. Negative control: undifferentiated ESCs. ∗p < 0.05 (n = 3 independent cultures, one pellet/culture mean ± SD).(D) Upregulation of N-cadherin surface expression on the FSB-expanded ectomesenchymal cells during FT treatment. The ectomesenchymal cells maintained under F (Fp2) and FSB (FSBp5) were either passaged to FSB (→FSB), FT (→FT, FTp2), or D10 (→D10) or not passaged (Fp2), and then FACS analyzed.(E) Effect of FT treatment on SOX9 protein expression. FSB-expanded p7 ectomesenchymal cells followed by FT culture (i.e., p8) were treated and analyzed as in Figure 3D. Graph, n = 4 technical repeats, mean ± SD. Isotype control, Figure S4J.(F) Real-time RT-PCR analysis for investigating the effect of FT treatment on SOX9 gene expression (n = 3 technical repeats, mean ± SD).
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fig4: Pretreatment with TGFβ Facilitates Cartilage Particle Formation from the FSB-Expanded Ectomesenchymal Cells(A and B) Cartilage particle formation from the H9 hESC-derived ectomesenchymal cells maintained under F (A) or FSB (B) by 3D-pellet culture. Toluidine Blue staining (purple) of sections of the cartilage particles, except for (Bvi), which is COL2 immunostained (brown). Some cultures were passaged once or twice (p2) to FT (CDM plus FGF2+TGFβ3) (Aiii, Bi, Bii, Bv, Bvi), or D10 (Aiv, Biii, Biv), prior to pellet culture.(C) Capacity of sGAG production in cartilage particles. The ectomesenchymal cells cultured as indicated (→FT, passage to FT) were subjected to pellet culture and total DNA and sGAGs were quantified. Negative control: undifferentiated ESCs. ∗p < 0.05 (n = 3 independent cultures, one pellet/culture mean ± SD).(D) Upregulation of N-cadherin surface expression on the FSB-expanded ectomesenchymal cells during FT treatment. The ectomesenchymal cells maintained under F (Fp2) and FSB (FSBp5) were either passaged to FSB (→FSB), FT (→FT, FTp2), or D10 (→D10) or not passaged (Fp2), and then FACS analyzed.(E) Effect of FT treatment on SOX9 protein expression. FSB-expanded p7 ectomesenchymal cells followed by FT culture (i.e., p8) were treated and analyzed as in Figure 3D. Graph, n = 4 technical repeats, mean ± SD. Isotype control, Figure S4J.(F) Real-time RT-PCR analysis for investigating the effect of FT treatment on SOX9 gene expression (n = 3 technical repeats, mean ± SD).
Mentions: The ectomesenchymal cells expanded in CDM in the presence of FGF2 or FGF2+SB431542 were subjected to 3D-pellet culture under the PDGF/TGFβ/BMP condition. The early (p2) FGF2-expanded cells formed a large cartilage particle that stained metachromatically (pink to purple) with Toluidine Blue, but then lost such chondrogenic activity by p4–p5 (Figures 4A and 4C), in keeping with the loss of the CD271+ cell population (Figures 2C and S3D).

Bottom Line: When "primed" with TGFβ, such cells efficiently formed translucent cartilage particles, which were completely mineralized in 12 weeks in immunocompromized mice.Ectomesenchyme is a source of many craniofacial bone and cartilage structures.The method we describe for obtaining a large quantity of human ectomesenchymal cells will help to model craniofacial disorders in vitro and potentially provide cells for the repair of craniofacial damage.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine, The University of Texas Health Science Center at Houston Medical School, Houston, TX 77030, USA.

Show MeSH
Related in: MedlinePlus