Fig 2: Expression of primed pluripotency cell-surface protein markers in bioreactor-cultured, naïve and primed hPSCs.a Average percentages of CD90-positive cells in bioreactor-cultured, naïve and primed H9 hPSCs. The data are represented as mean percentage of positive cells (n = 2). b Representative immunofluorescent images showing CD90 expression in bioreactor-cultured, naïve and primed H9 hPSC aggregates. CD90 antibody was conjugated with R-phycoerythrin (R-PE). Scale bars = 20 µm. c Histogram of flow cytometry analysis showing fluorescently labeled CD90, CD24, and CD317 in bioreactor-cultured, naïve and primed H9 hPSCs. Gates were drawn based on an isotype control. Aggregates of day 4 post-inoculation were used for flow cytometry analysis and for confocal imaging for both naïve and primed hPSCs. All the cultures underwent fed-batch condition (60% media change, 48-h post-inoculation) for bioreactor cultures. The data presented are generated from inoculation density of 50,000 cells/mL. d Expression dynamics of CD24 cell-surface protein during primed to naïve pluripotency conversion in the stirred suspension bioreactor within six days of culture. The media was changed gradually from mTESR1 to RSeT from day 2 to day 6 of culture in the bioreactor, and the cells were collected every day for analysis.

Fig 3: Gene expression and transcriptomic analyses of naïve and primed hPSCs.a Depiction of naïve- (KLF2, KLF4, STELLA, and KLF5) and primed- (CD24, OTX2, ZIC2, and DUSP6) hPSC-associated gene expression levels for H1 and H9 hESC lines analyzed by RT-qPCR. The aliquots of statically cultured, cells for each naïve (P4) and primed hPSC sample were collected for gene expression analysis before inoculating them into stirred suspension bioreactors. Those cells were counted as “NStat” and “PStat”. The aggregates of day 4 post-inoculation were collected for each naïve and primed hPSC sample for gene expression analysis. Expression was quantified relative to the housekeeping gene GAPDH and was normalized to statically cultured, primed hPSC (PStat) level (=1). All the cultures underwent fed-batch condition (60% media change, 48-h post-inoculation) for bioreactor cultures. The data presented are generated from inoculation density of 50,000 cells/mL. The data are represented as mean ± SEM (n = 3). **** = Adj-P < 0.0001, *** = Adj-P = 0.0002 and 0.0004, ** = Adj-P = 0.0021, * = Adj-P = 0.0291 using GraphPad Prism. Adj-P = Adjusted p value. RQ relative quantification. PStat = primed hPSCs cultured under static condition. PBior = primed hPSCs cultured under stirred suspension bioreactor condition. NStat = naïve hPSCs cultured under static condition. NBior = naïve hPSCs cultured under stirred suspension bioreactor condition. b RNA-sequencing analysis of naïve H9 hPSCs cultured under static suspension and static conditions. The violin plots show the enriched canonical pathways in static suspension compared to static culture (selected as a control reference) based on Ingenuity Pathway Analysis (IPA). The IPA z-score algorithm was used to identify enriched canonical pathways that are more active (positive z-score) or less active (negative z-score) according to the IPA database and observed gene expression in our RNA-seq dataset. Activated = genes expected to be upregulated (positive log2FC) if the pathway is activated according to IPA canonical pathway database. Inactivated = genes expected to be downregulated (negative log2FC) if the pathway is activated according to IPA canonical pathway database. No-expect = no direction of change expectation is available in the IPA canonical pathway database. Filter criteria for pathway enrichment significance was expected direction of change Z-score = 2 or =-2, and –log10 (Benjamini–Hochberg multiple testing corrected p value of differential expression) =2. The suspension culture underwent fed-batch condition (60% media change, 48-h post-inoculation). Aggregates of day 4 post-culture were collected for the analysis. The data are representative of three replicates.

Fig 4: Effect of FSTL1 knockdown on cell cycle and adhesion molecule expression in ccRCC cells. a Effect of FSTL1 knockdown on cell cycle progression of NRCC cells. The percentage of cells in S and G2/M phases is higher in NRCC-shFSTL1 cells (55.4% ± 4.0% and 8.8% ± 1.4%) than in NRCC-shScramble cells (39.0% ± 4.2% and 1.5% ± 0.8%). The first and second peaks of the red curve represent the cell count of G0/G1 and G2 phases, respectively. The shaded plot reflects the cell count of S phase. b, c Effect of FSTL1 knockdown on the expression of CD99 and CD24 on NRCC cells. The percentage of CD99-positive cells is higher in NRCC-shFSTL1 cells (44.7% ± 4.3%) than in NRCC-shScramble cells (15.3% ± 1.5%), whereas the percentage of CD24-positive cells is lower in NRCC-shFSTL1 cells than in NRCC-shScramble cells (55.5% ± 6.8% vs. 85.0% ± 1.2%). shFSTL1, FSTL1 knockdown; shScramle, vector control. Each flow cytometry assay was conducted in triplicate and a representative figure is shown

Fig 5: Emodin suppresses pulmonary metastasis of ovarian cancer by affecting the development of ovarian CSCs. (A) Representative images of the lungs from the three treatment groups and (B) numbers of metastatic nodules in the lungs were counted. (C) Weight of the lungs in each group was recorded at the end of the experiment. (D) The proportion of ovarian CSCs (CD44+/CD24-) in the lung tissues from each group was measured using flow cytometry and the results were (E) statistically analyzed. *P<0.05, **P<0.01, ***P<0.001 vs. control group (vehicle group).