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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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The SOX9-Expressing CD271+CD73+ Ectomesenchymal Cells Are Chondrogenic and Derived from the CD271hiCD73− Neural Crest-like Cell Fraction(A) Graphical representation of the experimental procedure.(B) FACS analysis of the sorted CD271hiCD73− (CD271hi) and CD271loCD73− (CD271lo) progeny, expanded under F, FSB, FSJN (FGF2+SJN2511), and FA83 (FGF2+A83-01) for three passages.(C) Real-time RT-PCR with cells in (B) demonstrating SOX9 expression specifically maintained in the FSB-expanded CD271hi cells. sort, pre-expanded cells (n = 3 technical repeats, mean ± SD).(D) Immunofluorescence detection of SOX9 protein in the FSB-expanded CD271hi cells. Pink, SOX9; blue (nucleus), DAPI; graph, the value of % SOX9+ nuclei was the average of scoring from four areas (n = 4 technical repeats, mean ± SD); ++, strong signal; +, weaker signal; isotype control, Figure S4J.(E) SOX9-GFP expression in the FSB-expanded CD271hi cells. SOX9-GFP hiPSCs were differentiated, and the CD271hiCD73− progeny were isolated and expanded under FSB for five passages as in (A) and then FACS analyzed (Figure S3H). H9 hESC-derived, FSB-expanded cells in (B) were used as the GFP-negative control.(F) Chondrogenic activity of the CD271hi or CD271lo progeny maintained under FSB. Cells were grown under FSB and F conditions for five passages and subjected to 2D-micromass cultures, followed by Alcian Blue staining and quantification. ∗p < 0.05 (n = 3 independent cultures, two masses/culture, mean ± SD).
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fig3: The SOX9-Expressing CD271+CD73+ Ectomesenchymal Cells Are Chondrogenic and Derived from the CD271hiCD73− Neural Crest-like Cell Fraction(A) Graphical representation of the experimental procedure.(B) FACS analysis of the sorted CD271hiCD73− (CD271hi) and CD271loCD73− (CD271lo) progeny, expanded under F, FSB, FSJN (FGF2+SJN2511), and FA83 (FGF2+A83-01) for three passages.(C) Real-time RT-PCR with cells in (B) demonstrating SOX9 expression specifically maintained in the FSB-expanded CD271hi cells. sort, pre-expanded cells (n = 3 technical repeats, mean ± SD).(D) Immunofluorescence detection of SOX9 protein in the FSB-expanded CD271hi cells. Pink, SOX9; blue (nucleus), DAPI; graph, the value of % SOX9+ nuclei was the average of scoring from four areas (n = 4 technical repeats, mean ± SD); ++, strong signal; +, weaker signal; isotype control, Figure S4J.(E) SOX9-GFP expression in the FSB-expanded CD271hi cells. SOX9-GFP hiPSCs were differentiated, and the CD271hiCD73− progeny were isolated and expanded under FSB for five passages as in (A) and then FACS analyzed (Figure S3H). H9 hESC-derived, FSB-expanded cells in (B) were used as the GFP-negative control.(F) Chondrogenic activity of the CD271hi or CD271lo progeny maintained under FSB. Cells were grown under FSB and F conditions for five passages and subjected to 2D-micromass cultures, followed by Alcian Blue staining and quantification. ∗p < 0.05 (n = 3 independent cultures, two masses/culture, mean ± SD).

Mentions: To determine the cellular origin of the CD271+CD73+ ectomesenchymal cells expanded under FGF2+SB431542 conditions, the CD271hiCD73− and CD271loCD73− progeny, developed from H9 hESCs and BJ5 hiPSCs, were sorted by FACS (Figures 1C and S1H) and then cultured in CDM in the presence of FGF2 and one of three Nodal/Activin/TGFβ inhibitors, SB431542, A83-01, or SJN2511. While FGF2 alone supported the accumulation of CD271−CD73+ cells from either progeny, the FGF2+SB431542, FGF2+A83-01, and FGF2+SJN2511 conditions preferentially supported the development of CD271+CD73+ cells from the sorted CD271hiCD73− neural crest-like progeny (Figures 3B and S4G). Concentrations of SB431542 between 1 and 3 μM were sufficient for the stable maintenance of CD271+CD73+ cells (Figure S3C). On the other hand, most of the CD271loCD73− progeny did not survive, even under FGF2+SB431542 conditions, and the derived mesenchymal cells were a mixture of CD271+CD73+ and CD271−CD73+ cells (Figures 3B and S4G) from which the CD271+CD73+ cells were later lost.


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 SOX9-Expressing CD271+CD73+ Ectomesenchymal Cells Are Chondrogenic and Derived from the CD271hiCD73− Neural Crest-like Cell Fraction(A) Graphical representation of the experimental procedure.(B) FACS analysis of the sorted CD271hiCD73− (CD271hi) and CD271loCD73− (CD271lo) progeny, expanded under F, FSB, FSJN (FGF2+SJN2511), and FA83 (FGF2+A83-01) for three passages.(C) Real-time RT-PCR with cells in (B) demonstrating SOX9 expression specifically maintained in the FSB-expanded CD271hi cells. sort, pre-expanded cells (n = 3 technical repeats, mean ± SD).(D) Immunofluorescence detection of SOX9 protein in the FSB-expanded CD271hi cells. Pink, SOX9; blue (nucleus), DAPI; graph, the value of % SOX9+ nuclei was the average of scoring from four areas (n = 4 technical repeats, mean ± SD); ++, strong signal; +, weaker signal; isotype control, Figure S4J.(E) SOX9-GFP expression in the FSB-expanded CD271hi cells. SOX9-GFP hiPSCs were differentiated, and the CD271hiCD73− progeny were isolated and expanded under FSB for five passages as in (A) and then FACS analyzed (Figure S3H). H9 hESC-derived, FSB-expanded cells in (B) were used as the GFP-negative control.(F) Chondrogenic activity of the CD271hi or CD271lo progeny maintained under FSB. Cells were grown under FSB and F conditions for five passages and subjected to 2D-micromass cultures, followed by Alcian Blue staining and quantification. ∗p < 0.05 (n = 3 independent cultures, two masses/culture, mean ± SD).
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fig3: The SOX9-Expressing CD271+CD73+ Ectomesenchymal Cells Are Chondrogenic and Derived from the CD271hiCD73− Neural Crest-like Cell Fraction(A) Graphical representation of the experimental procedure.(B) FACS analysis of the sorted CD271hiCD73− (CD271hi) and CD271loCD73− (CD271lo) progeny, expanded under F, FSB, FSJN (FGF2+SJN2511), and FA83 (FGF2+A83-01) for three passages.(C) Real-time RT-PCR with cells in (B) demonstrating SOX9 expression specifically maintained in the FSB-expanded CD271hi cells. sort, pre-expanded cells (n = 3 technical repeats, mean ± SD).(D) Immunofluorescence detection of SOX9 protein in the FSB-expanded CD271hi cells. Pink, SOX9; blue (nucleus), DAPI; graph, the value of % SOX9+ nuclei was the average of scoring from four areas (n = 4 technical repeats, mean ± SD); ++, strong signal; +, weaker signal; isotype control, Figure S4J.(E) SOX9-GFP expression in the FSB-expanded CD271hi cells. SOX9-GFP hiPSCs were differentiated, and the CD271hiCD73− progeny were isolated and expanded under FSB for five passages as in (A) and then FACS analyzed (Figure S3H). H9 hESC-derived, FSB-expanded cells in (B) were used as the GFP-negative control.(F) Chondrogenic activity of the CD271hi or CD271lo progeny maintained under FSB. Cells were grown under FSB and F conditions for five passages and subjected to 2D-micromass cultures, followed by Alcian Blue staining and quantification. ∗p < 0.05 (n = 3 independent cultures, two masses/culture, mean ± SD).
Mentions: To determine the cellular origin of the CD271+CD73+ ectomesenchymal cells expanded under FGF2+SB431542 conditions, the CD271hiCD73− and CD271loCD73− progeny, developed from H9 hESCs and BJ5 hiPSCs, were sorted by FACS (Figures 1C and S1H) and then cultured in CDM in the presence of FGF2 and one of three Nodal/Activin/TGFβ inhibitors, SB431542, A83-01, or SJN2511. While FGF2 alone supported the accumulation of CD271−CD73+ cells from either progeny, the FGF2+SB431542, FGF2+A83-01, and FGF2+SJN2511 conditions preferentially supported the development of CD271+CD73+ cells from the sorted CD271hiCD73− neural crest-like progeny (Figures 3B and S4G). Concentrations of SB431542 between 1 and 3 μM were sufficient for the stable maintenance of CD271+CD73+ cells (Figure S3C). On the other hand, most of the CD271loCD73− progeny did not survive, even under FGF2+SB431542 conditions, and the derived mesenchymal cells were a mixture of CD271+CD73+ and CD271−CD73+ cells (Figures 3B and S4G) from which the CD271+CD73+ cells were later lost.

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