Fig 2: Characterization of purified ES-MSCs derived from GDF6-treated H9 hESCs. The proportions of CD73+CD146+CD271+CD45– H9-MSC-V (a) and H9-MSC-G (b) cells were compared. c ALP staining and quantitative ALP activity assay in H9-MSC-V and H9-MSC-G cells after 14 days of OI. d ARS staining and quantification in H9-MSC-V and H9-MSC-G cells after 14 days of OI. e Alcian blue staining and quantification in H9-MSC-V and H9-MSC-G cells after 21 days of CI. f Oil Red O staining and quantification in H9-MSC-V and H9-MSC-G cells after 21 days of AI. Bar indicates 40 µm. qRT-PCR gene expression analysis of osteogenic (ALPL, RUNX2, IBSP, OCN) (g), chondrogenic (SOX9 and COL2a1) (h), and adipogenic markers (PPAR? and LPL) (i) after lineage-specific differentiation of H9-MSC-V and H9-MSC-G cells. j, k Bone formation in vivo by H9-MSC-V and H9-MSC-G in immunocompromised mice. Representative H&E staining of transplants (j) and quantitative analysis of mineralized tissue versus total area (k) are shown. n = 5. Bar indicates 40 µm. All in vitro experiments were performed three times independently. *P < 0.05, **P < 0.01, ***P < 0.001. For c–f and k, Student’s t test; for g–i, two-way ANOVA with Tukey’s post hoc test

Fig 3: SOX17-negative endothelium gives rise to SOX17-positive endothelium. (A) Experimental outline. After 2 days of differentiation as spin EBs, cells were harvested and plated in MC in a BL-CFC assay. After 1.75 days in MC, SOX17- ENDO cells were sorted and plated in a Matrigel endothelial tube assay. Six hours after seeding cells, time-lapse imaging of cells commenced in a humidified chamber at 37°C with 5% CO2 in air. EB, embryoid body; BL-CFC, blast colony-forming cell; MC, methylcellulose. (B) Gating strategy outlining the sorting of SOX17-ENDO cells from MC at d2+d1.75. The two left-hand panels show the expression of SOX17 and of CD34 at this time point. The right-hand panels show the sequential gating strategy used to sort the SOX17-ENDO (SOX17-CD34+CD43-CD73-) endothelial cells that were seeded into the endothelial tube assay. (C) Images taken from the time lapse series at illustrative time points from 0 to 3600 min as indicated on each image. Boxed areas and matching fields from intermediate time points are shown at higher magnification. The first faintly red fluorescent SOX17+ cells are seen from 360 min (6 h) and are obvious from 720 min (12 h) onwards. Blood cells appear from 2160 min (36 h). Scale bars: 100 µm. (D) Graph plotting the number of fluorescent SOX17 cells in the imaged field at hourly intervals, showing a rapid emergence of SOX17+ cells from 360 min to 1440 min, before a reduction and plateauing of numbers. The field area was 0.9 mm2. (E) Bar graph detailing the number of SOX17- and SOX17+ cells and the number of morphologically classified haematopoietic cells at the onset, and at 24 h and 48 h after the onset of time lapse imaging. There is little increase in cell number over the initial 24 h, although there is a rapid rise in the number of SOX17+ cells. See also Movie 1 and Fig. S2.

Fig 4: Effect of GDF6 treatment on the mesenchymal lineage commitment of H1 hESCs. a qRT-PCR analysis of the expression of mesodermal genes (FOXF1, MSX1, TBXT, KDR, GATA4) in H1 hESCs treated with 300 ng·mL-1 GDF6. b qRT-PCR analysis of the expression of MSC surface markers (CD73, CD146, and CD271) in H1 hESCs treated with 300 ng·mL-1 GDF6. c Flow cytometry analysis of CD73, CD146, and CD271 expression in vehicle- and GDF6-treated H1 cells. d ALP staining and quantitative ALP activity assay after 14 days of osteogenic induction (OI) for vehicle- or GDF6-treated H1 cells. e ARS staining and quantification after 14 days of OI for vehicle- or GDF6-treated H1 cells. f Alcian blue staining and quantification after 21 days of chondrogenic induction (CI) for vehicle- or GDF6-treated H1 cells. qRT-PCR gene expression analysis of osteogenic markers (ALPL, RUNX2, IBSP, OCN) (g) and chondrogenic markers (SOX9 and COL2a1) (h) after lineage-specific differentiation of H1 cells cultured in the presence or absence of GDF6. All in vitro experiments were performed three times independently. *P < 0.05, **P < 0.01, ***P < 0.001. For a–f, Student’s t test; for g and h, two-way ANOVA with Tukey’s post hoc test

Fig 5: Stem cell identification results. (a) Flow cytometry was used to detect the molecular markers of the third generation of hDPSCs, and the positive expression rates of CD45 were 0.34%, the positive expression rates of CD34 were 0.4%, the positive expression rates of CD90 were 99.7%, the positive expression rates of CD105 were 98.1%, and the positive expression rates of CD73 were 99.7%. (b) Third-passage hDPSCs were stained with Alizarin Red 28 days after osteogenic induction, and the results showed that a large number of mineralized nodules were formed. (c) Third-passage hDPSCs were stained with Oil Red O 21 days after the induction of adipogenesis, and the results showed lipid droplets forming. (d) The colony formation experiment results show the cells were cultured for 14 days, and the clonogenesis rate was 5-15 cells/1000 cells (number of cells = 50).