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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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In Vivo Stability of Cartilage Particles Developed with the CD271+CD73+ Ectomesenchymal Cells(A–D) In vivo maturation of cartilage particles developed with the FSB-expanded CD271+CD73+ ectomesenchymal cells (C and D), generated from H9 hESC-derived CD271hiCD73− neural crest-like progeny in comparison with those developed with the freshly isolated KDR−PDGFRα+ paraxial mesoderm, derived from H9 hESCs under the CHIR99021-based 4i+P conditions (A) and from HES3 hESCs under the BIO-based 2i condition (B) (Umeda et al., 2012). Cartilage particles were transplanted into NSG mice (A, C, and D) and NODScid mice (B) subcutaneously for 12 weeks, recovered, fixed, sectioned, and stained with Toluidine Blue (cartilaginous area is in purple) and von Kossa (bony area is in black).(E and F) Cartilage gene expression during chondrogenesis cultures. The FSB-expanded p7 cells were subjected to 2D-micromass culture (E) or 3D-pellet culture (F) and then real-time RT-PCR (n = 3 technical repeats, mean ± SD).(G and H) Immunostaining with anti-human mitochondria (hMit) antibody. Sections of a bony particle made in (C) and (D) were stained with H&E (G) or stained with hMit antibody (pink, G and H) and anti-type I collagen (COL1) antibody (green, H), followed by counterstaining with DAPI (blue). Negative controls, Figures S4K and S4L.
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fig7: In Vivo Stability of Cartilage Particles Developed with the CD271+CD73+ Ectomesenchymal Cells(A–D) In vivo maturation of cartilage particles developed with the FSB-expanded CD271+CD73+ ectomesenchymal cells (C and D), generated from H9 hESC-derived CD271hiCD73− neural crest-like progeny in comparison with those developed with the freshly isolated KDR−PDGFRα+ paraxial mesoderm, derived from H9 hESCs under the CHIR99021-based 4i+P conditions (A) and from HES3 hESCs under the BIO-based 2i condition (B) (Umeda et al., 2012). Cartilage particles were transplanted into NSG mice (A, C, and D) and NODScid mice (B) subcutaneously for 12 weeks, recovered, fixed, sectioned, and stained with Toluidine Blue (cartilaginous area is in purple) and von Kossa (bony area is in black).(E and F) Cartilage gene expression during chondrogenesis cultures. The FSB-expanded p7 cells were subjected to 2D-micromass culture (E) or 3D-pellet culture (F) and then real-time RT-PCR (n = 3 technical repeats, mean ± SD).(G and H) Immunostaining with anti-human mitochondria (hMit) antibody. Sections of a bony particle made in (C) and (D) were stained with H&E (G) or stained with hMit antibody (pink, G and H) and anti-type I collagen (COL1) antibody (green, H), followed by counterstaining with DAPI (blue). Negative controls, Figures S4K and S4L.

Mentions: Cartilage particles generated under standard “PDGF/TGFβ/BMP” conditions (Umeda et al., 2012) with the CD271+CD73+ ectomesenchymal cells, derived either from a mixture of the 6-day differentiated H9 hESCs or from the FACS-isolated CD271hiCD73− neural crest-like progeny (Figures 7C and 7D), were all mineralized after subcutaneous transplantation into immunocompromized mice for 8–12 weeks (black area with von Kossa staining), implying the capacity of the CD271+CD73+ cell to induce endochondral ossification. Consistently, the detection of RUNX2 transcript (2D-micromass culture, Figure 7E) and COL10A1 transcript (3D-pellet culture, Figure 7F) during chondrogenesis suggested that the ectomesenchymal cell-derived chondrocytes matured into hypertrophic chondrocytes.


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)

In Vivo Stability of Cartilage Particles Developed with the CD271+CD73+ Ectomesenchymal Cells(A–D) In vivo maturation of cartilage particles developed with the FSB-expanded CD271+CD73+ ectomesenchymal cells (C and D), generated from H9 hESC-derived CD271hiCD73− neural crest-like progeny in comparison with those developed with the freshly isolated KDR−PDGFRα+ paraxial mesoderm, derived from H9 hESCs under the CHIR99021-based 4i+P conditions (A) and from HES3 hESCs under the BIO-based 2i condition (B) (Umeda et al., 2012). Cartilage particles were transplanted into NSG mice (A, C, and D) and NODScid mice (B) subcutaneously for 12 weeks, recovered, fixed, sectioned, and stained with Toluidine Blue (cartilaginous area is in purple) and von Kossa (bony area is in black).(E and F) Cartilage gene expression during chondrogenesis cultures. The FSB-expanded p7 cells were subjected to 2D-micromass culture (E) or 3D-pellet culture (F) and then real-time RT-PCR (n = 3 technical repeats, mean ± SD).(G and H) Immunostaining with anti-human mitochondria (hMit) antibody. Sections of a bony particle made in (C) and (D) were stained with H&E (G) or stained with hMit antibody (pink, G and H) and anti-type I collagen (COL1) antibody (green, H), followed by counterstaining with DAPI (blue). Negative controls, Figures S4K and S4L.
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fig7: In Vivo Stability of Cartilage Particles Developed with the CD271+CD73+ Ectomesenchymal Cells(A–D) In vivo maturation of cartilage particles developed with the FSB-expanded CD271+CD73+ ectomesenchymal cells (C and D), generated from H9 hESC-derived CD271hiCD73− neural crest-like progeny in comparison with those developed with the freshly isolated KDR−PDGFRα+ paraxial mesoderm, derived from H9 hESCs under the CHIR99021-based 4i+P conditions (A) and from HES3 hESCs under the BIO-based 2i condition (B) (Umeda et al., 2012). Cartilage particles were transplanted into NSG mice (A, C, and D) and NODScid mice (B) subcutaneously for 12 weeks, recovered, fixed, sectioned, and stained with Toluidine Blue (cartilaginous area is in purple) and von Kossa (bony area is in black).(E and F) Cartilage gene expression during chondrogenesis cultures. The FSB-expanded p7 cells were subjected to 2D-micromass culture (E) or 3D-pellet culture (F) and then real-time RT-PCR (n = 3 technical repeats, mean ± SD).(G and H) Immunostaining with anti-human mitochondria (hMit) antibody. Sections of a bony particle made in (C) and (D) were stained with H&E (G) or stained with hMit antibody (pink, G and H) and anti-type I collagen (COL1) antibody (green, H), followed by counterstaining with DAPI (blue). Negative controls, Figures S4K and S4L.
Mentions: Cartilage particles generated under standard “PDGF/TGFβ/BMP” conditions (Umeda et al., 2012) with the CD271+CD73+ ectomesenchymal cells, derived either from a mixture of the 6-day differentiated H9 hESCs or from the FACS-isolated CD271hiCD73− neural crest-like progeny (Figures 7C and 7D), were all mineralized after subcutaneous transplantation into immunocompromized mice for 8–12 weeks (black area with von Kossa staining), implying the capacity of the CD271+CD73+ cell to induce endochondral ossification. Consistently, the detection of RUNX2 transcript (2D-micromass culture, Figure 7E) and COL10A1 transcript (3D-pellet culture, Figure 7F) during chondrogenesis suggested that the ectomesenchymal cell-derived chondrocytes matured into hypertrophic chondrocytes.

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