Fig 2: Characterization of purified ES-MSCs derived from GDF6-treated H1 hESCs. Proportions of CD73+CD146+CD271+CD45- H1-MSC-V (a) and H1-MSC-G (b) were compared by FACS. c ALP staining and quantitative ALP activity assay in H1-MSC-V and H1-MSC-G cells after 14 days of OI. d ARS staining and quantification in H1-MSC-V and H1-MSC-G cells after 14 days of OI. e Alcian blue staining and quantification in H1-MSC-V and H1-MSC-G cells after 21 days of CI. f Oil Red O staining and quantification after 21 days of adipogenic induction (AI) in H1-MSC-V and H1-MSC-G cells. 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 in H1-MSC-V and H1-MSC-G cells. j, k In vivo bone formation of H1-MSC-V and H1-MSC-G cells in immunocompromised mice. Representative H&E staining of transplants (j) and quantitative analysis of mineralized tissue versus total area (k). 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: 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 4: Determination of T Cell Receptor (TCR) Genes for a Corresponding Neoantigen-Specific T Cell by Using NP-Barcoded NACS(A) The optical micrograph shows a captured barcoded T cell from patient #1, followed by the 3 sequential fluorescent readout steps to identify specificity against neoantigen 12. Scale bars represent 20 µm. The captured single T cell was punched out for RT-PCR to obtain TCRa and TCRß gene sequences (DNA ladder: 100 bp). The TCR gene was then assembled and inserted into a retroviral vector and transduced into Jurkat T cells for analysis by flow cytometry.(B) Flow cytometry results for untransduced Jurkats (left), Jurkats transduced with LNGFR expression reporter only (center), and Jurkats transduced with both LNGFR and the TCR specific for neoantigen 12 (right).See also Figures S3 and S4.

Fig 5: Effect of GSK126 Treatment on Mesenchymal Lineage Commitment of H1 hESCs(A) qRT-PCR gene expression analysis of well-known MSC surface markers (CD73, CD146, and CD271).(B) Flow-cytometry analysis for CD73, CD146, and CD271 expression of cells treated with DMSO or GSK126.(C) ALP staining and quantitative ALP activity assay after 14 days of osteogenic induction (OI) for DMSO- or GSK126-treated cells. Scale bar, 300 µm.(D) ARS staining and quantification after 14 days of OI for DMSO- or GSK126-treated cells. Scale bar, 440 µm.(E) Alcian blue staining and quantification after 21 days of chondrogenic induction (CI) for DMSO- or GSK126-treated cells. Scale bar, 440 µm.(F and G) qRT-PCR gene expression analysis of osteogenic markers (ALPL, RUNX2, IBSP, and BGLAP) (F) and chondrogenic markers (SOX9 and COL2a1) (G) after lineage-specific differentiation in H1 cells treated with or without GSK126.(H) Proportions of CD90+CD146+CD271+CD45– H1-MSC-V and H1-MSC-126 are compared.(I) ALP staining and quantitative ALP activity assay of H1-MSC-V and H1-MSC-126 after 14 days of OI. Scale bar, 440 µm.(J) ARS staining and quantification of H1-MSC-V and H1-MSC-126 after 14 days of OI. Scale bar, 440 µm.(K) qRT-PCR gene expression analysis of osteogenic markers (ALPL, RUNX2, IBSP, and BGLAP) in H1-MSC-V and H1-MSC-126 after 14 days of OI.(L) Alcian blue staining and quantification (left) and qRT-PCR gene expression analysis of chondrogenic markers (SOX9 and COL2a1) (right) of H1-MSC-V and H1-MSC-126 after 21 days of CI. Scale bar, 440 µm.(M) Oil red O staining and quantification (left) and qRT-PCR gene expression analysis of adipogenic markers (PPARG and LPL) (right) of H1-MSC-V and H1-MSC-126 after 21 days of adipogenic induction (AI). Scale bar, 440 µm.Data are shown as mean ± SD; n = 3 independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 by Student's t test. See also Figures S3 and S4.