Fig 2: Characterizing differential gene expression among CTCs, primary tumor, and HCC cell lines based on RNA-seq.a Overview of the workflow for CTC isolation and single-cell RNA preparation. b Representative fluorescence images of CTCs and WBCs labeled with CD45, EpCAM, and CK antibodies; the scale bar is 10 μm. c CNV profiling of seven EpCAM+ and/or pan-CK+ and CD45− CTCs and two CD45+ WBC from six patients. The fluorescence images of sequenced CTCs and WBCs are displayed. d t-SNE plot illustrates the similarity of the expression profiles between CTCs, primary tumors, and HCC cell lines. e Pathway enrichment in the Hallmark dataset for upregulated genes in CTCs compared with primary tumors. Hypergeometric test was used to test whether DEGs were overlapped with the gene sets. The Benjamini–Hochberg FDR-controlling method for multiple hypothesis testing were performed.

Fig 3: Demonstration of continuous transfer of exogenous mitochondria in blood of Polg mouse model.Lineage negative cells ubiquitously expressing red-labeled nucleus (dTomato) and green-labeled mitochondria (Dendra) derived from ROSAnT-nG/PhAMexcised mice were augmented with Dendra2+ mitochondria for 21 h and injected to Polg mice dosed at age 24–27 weeks (males) and 30-34 weeks (females). At 12 h, 1 w, 1 m, and 4.5 m post-injection blood was drawn and BM was extracted and both were analyzed by flow cytometry and confocal microscopy. a Graphical presentation of experimental design and a representative image of confocal microscopy of dTomato+Dendra2+ (left) and dTomato-Dendra2+ (right) found in bone marrow 1 m post-injection. Scale bar 5 µm. b Representation of flow cytometry analysis, which is expected to detect three types of cells: Polg cells, transplanted/daughter cells (red box, dTomato+Dendra2+) and Polg cells which received mitochondrial transfer (green box, dTomato-Dendra2+). c Representative figure of flow cytometry analysis of dTomato-Dendra2+ cells within CD45+ cell population in peripheral blood 12 h, 1 w, 1 m and 4.5 m post-injection (left). Percentages of dTomato+Dendra2+ cells and dTomato-Dendra2+ cells in peripheral blood (right). d Percentages of CD3+ T cells, CD19+ B cells and CD11b+ myeloid cells within the CD45+ and dTomato-Dendra2+ populations in peripheral blood 1 m post-treatment. e Percentages of dTomato+Dendra2+ cells (left) and dTomato-Dendra2+ cells (right) in the bone marrow. f Representative image of confocal microscopy analysis of BM extracted cells at 4.5 months after treatment, demonstrating Dendra2+ mitochondria transfer between adjacent cells. Left, differential interference contrast (DIC); center, merged florescent image from DAPI, Dendra2 and dTomato channels. Scale bar, 5 µm; right, 3D reconstruction of the Z-stack images. BM bone marrow, HSPCs hematopoietic stem and progenitor cells.

Fig 4: WNT inhibition during pellet culture enhanced homogeneity of hiPSC chondrogenesis.A Experimental scheme of WNT inhibition. B C59 treatment during pellet culture enhanced Saf-O staining and decreased off-target cells (yellow arrowheads) as compared to other WNT inhibition culture regiments. Top row scale bar = 400 µm. Bottom row scale bar = 200 µm. The experiment was performed twice with similar results. C Pellets treated with C59 in only pellet culture exhibited an increased GAG/DNA ratio compared to pellets treated with other culture regiments. *p = 0.00001 at d28. #p = 0.0228 at d42. Mean ± SEM. n = 4 pellets per group. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test at a specific time point. D C59 significantly decreased, but WNT3A significantly increased, CD146+/CD166+/CD45- progenitors at the Cp stage. Different letters are significantly different (a vs. b, p = 0.0005; a vs. c, p = 0.0021; b vs. c, p = 0.0001). Mean ± SEM. n = 3 per group (independent experiment). Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. E RNA-FISH of d28 pellets showing C59-treated pellets had decreased WNT3A and WNT4 labeling (green) but more homogenous COL2A1 distribution (red) in the pellets. Scale bar = 200 µm. The experiment was performed twice with similar results.

Fig 5: Immunophenotypes of A-MSCs and HUVECs as determined by flow cytometry. (A) Immunophenotype of monolayer and 3D spheroid A-MSCs. The expression of positive stem markers CD29, CD44, CD73, CD90, and CD105 and negative stem markers CD31, CD45, and CD271. (B) Phenotypic identification of HUVECs. The isolated cultured cells were stained with endothelial cell marker CD31. A-MSCs: Adipose-derived mesenchymal stem cells; HUVECs: human umbilical vein endothelial cells.