Limits...
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.

Show MeSH

Related in: MedlinePlus

The Long-Term Expanded Ectomesenchymal Cells Are Still Dependent on FSB(A) SB431542-dependent maintenance of CD271 expression. Ectomesenchymal cells maintained under FSB (p10) were either passaged one to three times to FSB again (p11, p14), or to FGF2 (→F, Fp3) and then FACS analyzed.(B) Maintenance of sGAG production capacity. The ectomesenchymal cells maintained under FSB for 11–16 passages followed by FT treatment, and the freshly isolated chondrogenic paraxial mesoderm (PM) (Umeda et al., 2012) were subjected to pellet culture. The capacity for sGAG production is displayed as in Figure 4C. Negative control, hESCs; positive control, bovine articular cartilage (n = 3 technical repeats, mean ± SD).(C) “Full-cartilage”-forming capacity remained in the FSB-expanded p13 ectomesenchymal cells derived from CD271hiCD73− H9 progeny. Toluidine Blue staining.(D) SB431542-dependent maintenance of chondrogenic activity. The p10 ectomesenchymal cells were passaged to either FSB for four passages (FSBp14) or F for two passages (→Fp2) and then subjected to FT treatment and 3D-pellet culture. The capacity for sGAG production is displayed as in (B). ∗p < 0.05 (n ≥ 3 independent cultures, one pellet/culture, mean ± SD).(E) Real-time RT-PCR analysis for investigating the effect of FSB on maintaining SOX9 expression (n = 3 technical repeats, mean ± SD).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4400647&req=5

fig5: The Long-Term Expanded Ectomesenchymal Cells Are Still Dependent on FSB(A) SB431542-dependent maintenance of CD271 expression. Ectomesenchymal cells maintained under FSB (p10) were either passaged one to three times to FSB again (p11, p14), or to FGF2 (→F, Fp3) and then FACS analyzed.(B) Maintenance of sGAG production capacity. The ectomesenchymal cells maintained under FSB for 11–16 passages followed by FT treatment, and the freshly isolated chondrogenic paraxial mesoderm (PM) (Umeda et al., 2012) were subjected to pellet culture. The capacity for sGAG production is displayed as in Figure 4C. Negative control, hESCs; positive control, bovine articular cartilage (n = 3 technical repeats, mean ± SD).(C) “Full-cartilage”-forming capacity remained in the FSB-expanded p13 ectomesenchymal cells derived from CD271hiCD73− H9 progeny. Toluidine Blue staining.(D) SB431542-dependent maintenance of chondrogenic activity. The p10 ectomesenchymal cells were passaged to either FSB for four passages (FSBp14) or F for two passages (→Fp2) and then subjected to FT treatment and 3D-pellet culture. The capacity for sGAG production is displayed as in (B). ∗p < 0.05 (n ≥ 3 independent cultures, one pellet/culture, mean ± SD).(E) Real-time RT-PCR analysis for investigating the effect of FSB on maintaining SOX9 expression (n = 3 technical repeats, mean ± SD).

Mentions: Since CD271+CD73+ ectomesenchymal cells maintained under FGF2+SB431542 for a long time might select a rare “permanent CD271+ chondrogenic cell” from the original neural crest cell population, late-passage (p10) cells were subjected to medium change to CDM with FGF2 alone. All cells started losing the CD271 expression in FGF2 and nearly 95% became CD271−CD73+ by passage 3 (Figure 5A). Those maintained in FGF2+SB431542 for four more passages were still CD271+CD73+, suggesting that maintenance of CD271 surface expression remained dependent on the presence of SB431542.


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)

The Long-Term Expanded Ectomesenchymal Cells Are Still Dependent on FSB(A) SB431542-dependent maintenance of CD271 expression. Ectomesenchymal cells maintained under FSB (p10) were either passaged one to three times to FSB again (p11, p14), or to FGF2 (→F, Fp3) and then FACS analyzed.(B) Maintenance of sGAG production capacity. The ectomesenchymal cells maintained under FSB for 11–16 passages followed by FT treatment, and the freshly isolated chondrogenic paraxial mesoderm (PM) (Umeda et al., 2012) were subjected to pellet culture. The capacity for sGAG production is displayed as in Figure 4C. Negative control, hESCs; positive control, bovine articular cartilage (n = 3 technical repeats, mean ± SD).(C) “Full-cartilage”-forming capacity remained in the FSB-expanded p13 ectomesenchymal cells derived from CD271hiCD73− H9 progeny. Toluidine Blue staining.(D) SB431542-dependent maintenance of chondrogenic activity. The p10 ectomesenchymal cells were passaged to either FSB for four passages (FSBp14) or F for two passages (→Fp2) and then subjected to FT treatment and 3D-pellet culture. The capacity for sGAG production is displayed as in (B). ∗p < 0.05 (n ≥ 3 independent cultures, one pellet/culture, mean ± SD).(E) Real-time RT-PCR analysis for investigating the effect of FSB on maintaining SOX9 expression (n = 3 technical repeats, mean ± SD).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400647&req=5

fig5: The Long-Term Expanded Ectomesenchymal Cells Are Still Dependent on FSB(A) SB431542-dependent maintenance of CD271 expression. Ectomesenchymal cells maintained under FSB (p10) were either passaged one to three times to FSB again (p11, p14), or to FGF2 (→F, Fp3) and then FACS analyzed.(B) Maintenance of sGAG production capacity. The ectomesenchymal cells maintained under FSB for 11–16 passages followed by FT treatment, and the freshly isolated chondrogenic paraxial mesoderm (PM) (Umeda et al., 2012) were subjected to pellet culture. The capacity for sGAG production is displayed as in Figure 4C. Negative control, hESCs; positive control, bovine articular cartilage (n = 3 technical repeats, mean ± SD).(C) “Full-cartilage”-forming capacity remained in the FSB-expanded p13 ectomesenchymal cells derived from CD271hiCD73− H9 progeny. Toluidine Blue staining.(D) SB431542-dependent maintenance of chondrogenic activity. The p10 ectomesenchymal cells were passaged to either FSB for four passages (FSBp14) or F for two passages (→Fp2) and then subjected to FT treatment and 3D-pellet culture. The capacity for sGAG production is displayed as in (B). ∗p < 0.05 (n ≥ 3 independent cultures, one pellet/culture, mean ± SD).(E) Real-time RT-PCR analysis for investigating the effect of FSB on maintaining SOX9 expression (n = 3 technical repeats, mean ± SD).
Mentions: Since CD271+CD73+ ectomesenchymal cells maintained under FGF2+SB431542 for a long time might select a rare “permanent CD271+ chondrogenic cell” from the original neural crest cell population, late-passage (p10) cells were subjected to medium change to CDM with FGF2 alone. All cells started losing the CD271 expression in FGF2 and nearly 95% became CD271−CD73+ by passage 3 (Figure 5A). Those maintained in FGF2+SB431542 for four more passages were still CD271+CD73+, suggesting that maintenance of CD271 surface expression remained dependent on the presence of SB431542.

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