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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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FGF2+SB431542 Generates and Expands SOX9-Expressing CD271+PDGFRα+CD73+ Ectomesenchymal Cells from Unsorted Nonmesendodermal Progeny(A) Graphical representation of the experimental procedure. SB, SB431542; F, FGF2; FSB, FGF2+SB431542.(B) Growth curve of H9 hESC-derived ectomesenchymal cells expanded under F, FSB, and SB conditions (n = 3 independent cultures, mean ± SD).(C) FACS analysis demonstrating the effect of FSB on the selective accumulation of CD271+CD73+ mesenchymal. H9 hESCs and BJ5 hiPSCs were differentiated (Pre) and expanded under the conditions indicated: D10, F, FSB, and FLDN (FGF2+LDN193189). p, passage; isotype control, Figure S3C.(D) Micrographs of mesenchymal cells (p4) expanded under F and FSB.(E) Real-time RT-PCR analysis demonstrating changes in the neural crest gene expression during expansion under F and FSB (n = 3 technical repeats, mean ± SD).
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fig2: FGF2+SB431542 Generates and Expands SOX9-Expressing CD271+PDGFRα+CD73+ Ectomesenchymal Cells from Unsorted Nonmesendodermal Progeny(A) Graphical representation of the experimental procedure. SB, SB431542; F, FGF2; FSB, FGF2+SB431542.(B) Growth curve of H9 hESC-derived ectomesenchymal cells expanded under F, FSB, and SB conditions (n = 3 independent cultures, mean ± SD).(C) FACS analysis demonstrating the effect of FSB on the selective accumulation of CD271+CD73+ mesenchymal. H9 hESCs and BJ5 hiPSCs were differentiated (Pre) and expanded under the conditions indicated: D10, F, FSB, and FLDN (FGF2+LDN193189). p, passage; isotype control, Figure S3C.(D) Micrographs of mesenchymal cells (p4) expanded under F and FSB.(E) Real-time RT-PCR analysis demonstrating changes in the neural crest gene expression during expansion under F and FSB (n = 3 technical repeats, mean ± SD).

Mentions: Either in a FACS-purified form or in an unpurified mixture with other nonmesendodermal (i.e., MIXL1−) cells, the CD271hiCD73− neural crest-like progeny failed to adhere to the culture dish in the absence of fibronectin and grew poorly in the medium in which they were specified, i.e., CDM plus SB431542 (SB; Figures 2B and S3A). Therefore, we tested the effects of growth factors, such as FGF2 that have been used for maintaining neural crest cells (Stemple and Anderson, 1992) and generating chondrogenic activity (Abzhanov et al., 2003) in culture, and of other factors, such as endothelin1, PDGF, and Sonic hedgehog that have been linked to cranial skeletogenesis and ectomesenchymal specification in vivo (Le Douarin and Creuzet, 2009). FGF2 alone significantly increased viability and supported growth of the neural crest-like progeny on fibronectin but was often associated with significant slowdown in an early stage of expansion culture. The addition of both FGF2 and SB431542 maintained the growth rate at least until p16 and supported maintenance of the normal karyotype at least to p10 (Figure S3B).


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)

FGF2+SB431542 Generates and Expands SOX9-Expressing CD271+PDGFRα+CD73+ Ectomesenchymal Cells from Unsorted Nonmesendodermal Progeny(A) Graphical representation of the experimental procedure. SB, SB431542; F, FGF2; FSB, FGF2+SB431542.(B) Growth curve of H9 hESC-derived ectomesenchymal cells expanded under F, FSB, and SB conditions (n = 3 independent cultures, mean ± SD).(C) FACS analysis demonstrating the effect of FSB on the selective accumulation of CD271+CD73+ mesenchymal. H9 hESCs and BJ5 hiPSCs were differentiated (Pre) and expanded under the conditions indicated: D10, F, FSB, and FLDN (FGF2+LDN193189). p, passage; isotype control, Figure S3C.(D) Micrographs of mesenchymal cells (p4) expanded under F and FSB.(E) Real-time RT-PCR analysis demonstrating changes in the neural crest gene expression during expansion under F and FSB (n = 3 technical repeats, mean ± SD).
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fig2: FGF2+SB431542 Generates and Expands SOX9-Expressing CD271+PDGFRα+CD73+ Ectomesenchymal Cells from Unsorted Nonmesendodermal Progeny(A) Graphical representation of the experimental procedure. SB, SB431542; F, FGF2; FSB, FGF2+SB431542.(B) Growth curve of H9 hESC-derived ectomesenchymal cells expanded under F, FSB, and SB conditions (n = 3 independent cultures, mean ± SD).(C) FACS analysis demonstrating the effect of FSB on the selective accumulation of CD271+CD73+ mesenchymal. H9 hESCs and BJ5 hiPSCs were differentiated (Pre) and expanded under the conditions indicated: D10, F, FSB, and FLDN (FGF2+LDN193189). p, passage; isotype control, Figure S3C.(D) Micrographs of mesenchymal cells (p4) expanded under F and FSB.(E) Real-time RT-PCR analysis demonstrating changes in the neural crest gene expression during expansion under F and FSB (n = 3 technical repeats, mean ± SD).
Mentions: Either in a FACS-purified form or in an unpurified mixture with other nonmesendodermal (i.e., MIXL1−) cells, the CD271hiCD73− neural crest-like progeny failed to adhere to the culture dish in the absence of fibronectin and grew poorly in the medium in which they were specified, i.e., CDM plus SB431542 (SB; Figures 2B and S3A). Therefore, we tested the effects of growth factors, such as FGF2 that have been used for maintaining neural crest cells (Stemple and Anderson, 1992) and generating chondrogenic activity (Abzhanov et al., 2003) in culture, and of other factors, such as endothelin1, PDGF, and Sonic hedgehog that have been linked to cranial skeletogenesis and ectomesenchymal specification in vivo (Le Douarin and Creuzet, 2009). FGF2 alone significantly increased viability and supported growth of the neural crest-like progeny on fibronectin but was often associated with significant slowdown in an early stage of expansion culture. The addition of both FGF2 and SB431542 maintained the growth rate at least until p16 and supported maintenance of the normal karyotype at least to p10 (Figure S3B).

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