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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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Directed Specification of Neural Crest from hPSCs(A) FACS analysis demonstrating the SB431542-dependent development of CD271hiPDGFRαlo progeny during 2D differentiation of H9 and MIXL1-GFP hESCs in CDM. SB: 10 μM SB431542.(B) Time-dependent changes in the gene expression profile during differentiation of H9 hESCs in the presence of SB (n = 3 technical repeats, mean ± SD).(C) Isolation of H9-derived CD271hi(PDGFRαloCD73−) and CD271lo(PDGFRα−CD73−) progeny by FACS. (Left) Isotype control.(D) Real-time RT-PCR analysis with cells from (C) demonstrating enrichment of neural crest-related transcripts in the CD271hiCD73− cell fraction. Pre, presort cells (n = 3 technical repeats, mean ± SD).(E) Intracellular FACS staining by anti-PAX3 antibody demonstrating presence of PAX3 protein in the H9-derived CD271hiCD73− cell fraction. (Upper) Isotype control.(F) FACS demonstration of SOX9-GFP expression in the CD271hiCD73− progeny in SOX9-GFP hiPSCs differentiated for 6 days.
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fig1: Directed Specification of Neural Crest from hPSCs(A) FACS analysis demonstrating the SB431542-dependent development of CD271hiPDGFRαlo progeny during 2D differentiation of H9 and MIXL1-GFP hESCs in CDM. SB: 10 μM SB431542.(B) Time-dependent changes in the gene expression profile during differentiation of H9 hESCs in the presence of SB (n = 3 technical repeats, mean ± SD).(C) Isolation of H9-derived CD271hi(PDGFRαloCD73−) and CD271lo(PDGFRα−CD73−) progeny by FACS. (Left) Isotype control.(D) Real-time RT-PCR analysis with cells from (C) demonstrating enrichment of neural crest-related transcripts in the CD271hiCD73− cell fraction. Pre, presort cells (n = 3 technical repeats, mean ± SD).(E) Intracellular FACS staining by anti-PAX3 antibody demonstrating presence of PAX3 protein in the H9-derived CD271hiCD73− cell fraction. (Upper) Isotype control.(F) FACS demonstration of SOX9-GFP expression in the CD271hiCD73− progeny in SOX9-GFP hiPSCs differentiated for 6 days.

Mentions: Generation of osteochondrogenic ectomesenchyme from hPSCs first requires specification of cranial neural crest-like progeny. In order to reproducibly generate strong chondrogenic activity from hPSCs, we first optimized the method for specification of neural crest using CD271 as the readout. Fluorescence-activated cell sorting (FACS) analyses revealed that when H9 hESCs and BJ5 hiPSCs were differentiated in CDM using the conventional embryoid body (EB)-forming culture or 2D differentiation culture in the presence of SB431542 or other Nodal/Activin/TGFβ signaling inhibitor (e.g., A83-01 or SJN2511), they generated progeny that expressed CD271 (Figures 1A and S1). The CD271hi cell population, but not the CD271lo/− progeny, also expressed PDGFRα at low levels. Both populations lacked CD73 and CD13. The effect of SB431542 was dose dependent at least up to 10 μM (Figure S1D).


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)

Directed Specification of Neural Crest from hPSCs(A) FACS analysis demonstrating the SB431542-dependent development of CD271hiPDGFRαlo progeny during 2D differentiation of H9 and MIXL1-GFP hESCs in CDM. SB: 10 μM SB431542.(B) Time-dependent changes in the gene expression profile during differentiation of H9 hESCs in the presence of SB (n = 3 technical repeats, mean ± SD).(C) Isolation of H9-derived CD271hi(PDGFRαloCD73−) and CD271lo(PDGFRα−CD73−) progeny by FACS. (Left) Isotype control.(D) Real-time RT-PCR analysis with cells from (C) demonstrating enrichment of neural crest-related transcripts in the CD271hiCD73− cell fraction. Pre, presort cells (n = 3 technical repeats, mean ± SD).(E) Intracellular FACS staining by anti-PAX3 antibody demonstrating presence of PAX3 protein in the H9-derived CD271hiCD73− cell fraction. (Upper) Isotype control.(F) FACS demonstration of SOX9-GFP expression in the CD271hiCD73− progeny in SOX9-GFP hiPSCs differentiated for 6 days.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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fig1: Directed Specification of Neural Crest from hPSCs(A) FACS analysis demonstrating the SB431542-dependent development of CD271hiPDGFRαlo progeny during 2D differentiation of H9 and MIXL1-GFP hESCs in CDM. SB: 10 μM SB431542.(B) Time-dependent changes in the gene expression profile during differentiation of H9 hESCs in the presence of SB (n = 3 technical repeats, mean ± SD).(C) Isolation of H9-derived CD271hi(PDGFRαloCD73−) and CD271lo(PDGFRα−CD73−) progeny by FACS. (Left) Isotype control.(D) Real-time RT-PCR analysis with cells from (C) demonstrating enrichment of neural crest-related transcripts in the CD271hiCD73− cell fraction. Pre, presort cells (n = 3 technical repeats, mean ± SD).(E) Intracellular FACS staining by anti-PAX3 antibody demonstrating presence of PAX3 protein in the H9-derived CD271hiCD73− cell fraction. (Upper) Isotype control.(F) FACS demonstration of SOX9-GFP expression in the CD271hiCD73− progeny in SOX9-GFP hiPSCs differentiated for 6 days.
Mentions: Generation of osteochondrogenic ectomesenchyme from hPSCs first requires specification of cranial neural crest-like progeny. In order to reproducibly generate strong chondrogenic activity from hPSCs, we first optimized the method for specification of neural crest using CD271 as the readout. Fluorescence-activated cell sorting (FACS) analyses revealed that when H9 hESCs and BJ5 hiPSCs were differentiated in CDM using the conventional embryoid body (EB)-forming culture or 2D differentiation culture in the presence of SB431542 or other Nodal/Activin/TGFβ signaling inhibitor (e.g., A83-01 or SJN2511), they generated progeny that expressed CD271 (Figures 1A and S1). The CD271hi cell population, but not the CD271lo/− progeny, also expressed PDGFRα at low levels. Both populations lacked CD73 and CD13. The effect of SB431542 was dose dependent at least up to 10 μM (Figure S1D).

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