Figure 3

Prospective isolation of paraxial mesoderm and demonstration of their chondrogenic activity.

(A, B) Enrichment of paraxial mesoderm generated from H9 hES cells. H9 hES cells were differentiated under BION conditions and total progeny (Pre) and the KDR⁻PDGFRα⁺ (P+, red gate in A) and KDR⁻PDGFRα⁻ (P-, blue gate in A) progeny were isolated by FACS and subjected to RT-PCR (B). (C) MEOX1 protein expression. Mesodermal progeny, derived from H9 hES cells in the presence of BIO + Noggin (BION) or BIO alone (BIO) and cultured on a chamber slide for 5 day, were immunostained with the anti-MEOX1 antibody. The nucleoli staining pink within a blue nucleus, some of which are indicated with white arrows, are detectable in progeny generated under the BION condition. Isotype controls are shown in Supplementary Fig. 1C. Scale Bars, 50 μm. (D) Time-course of FACS analysis for MIXL1-GFP (GFP), KDR and PDGFRα expression during Mixl1-GFP hES cell differentiation induced in the presence of BIO + SB + Noggin (BIOSN). (E, F) Enrichment of paraxial mesoderm derived from other hPS cells. Mixl1-GFP hES cells and HK1 hiPS cells were differentiated under BIOSN conditions. From Mixl1-GFP hES cells, total EB cells (Pre) and the GFP⁺KDR⁻PDGFRα⁺ (GFP+P+ red gate) and GFP⁺KDR⁻PDGFRα⁻ (GFP+P− blue gate) progeny were isolated by FACS (E) and subjected to RT-PCR using the MEOX1 primers (F). From HK1 hiPS cells, only the KDR⁻PDGFRα⁺ (P+) and KDR⁻PDGFRα⁻ (P−) progeny were accumulated (E) as in the case of H9 hES cells.