Journal: Stem Cell Research & Therapy

Article Title: Good manufacturing practice production of human corneal limbus-derived stromal stem cells and in vitro quality screening for therapeutic inhibition of corneal scarring

doi: 10.1186/s13287-023-03626-8

Figure Lengend Snippet: Comparative assessment of cryopreservation media for cultured CSSCs. A Cell attachment efficiency—thawed cells after one month of frozen storage in different cryopreservation media was seeded on hFn-coated culture surface. After 24 h, the attachment rates of cells stored in CryoStor DMSO 5% and Cryopres DMSO 5% were similar to the research-grade DMSO [Res] (5%). Cell frozen in DMSO-free Stem-CellBanker, CryoStor DMSO 10%, and Cryopres DMSO 10% had poor cell attachment. B Cell index profiles of thawed CSSCs by xCELLigence. Cells in CryoStor DMSO 5% exhibited better growth kinetics. C Apoptosis assay by Annexin V-PI staining showed the percentages of live cells after frozen storage in 5% DMSO [Res] , CryoStor DMSO, 5% and Cryopres DMSO 5% were similar

Article Snippet: After allowing to attach for 24 h, the cells were differentiated to keratocytes by incubating with DMEM containing GlutaMAX I, 1 g/L D-glucose and sodium pyruvate (Thermo Fisher), ascorbate-2-phosphate (1 mM; Sigma), bFGF (10 ng/ml, Gibco), and transforming growth factor β3 (TGFβ3, 0.1 ng/ml; Gibco) [ ], and replenished with fresh medium every 2 to 3 days.

Techniques: Cell Culture, Cell Attachment Assay, Apoptosis Assay, Staining

Journal: eLife

Article Title: Skeletal dysplasia-causing TRPV4 mutations suppress the hypertrophic differentiation of human iPSC-derived chondrocytes

doi: 10.7554/eLife.71154

Figure Lengend Snippet: Moderate V620I and severe T89I mutations have distinct gene expression after 28 and 56 days of chondrogenic differentiation with TGFβ3. ( A ) A heatmap representing the log 2 fold change, compared to wildtype (WT), of the top 15 most up- and down-regulated genes unique to V620I and T89I at day 28. ( B ) The expression of protein kinase C alpha ( PRKCA ) at day 28 for WT, V620I, and T89I represented by normalized counts. *p adj ≤ 0.1 and log 2 (fold change) ≥1 (i.e., differentially expressed). ( C ) A heatmap representing the log 2 fold change, compared to WT, of the top 15 most up- and down-regulated genes unique to V620I and T89I at day 56. ( D ) The expression of catenin beta 1 ( CTNNB1 ) at day 56 for WT, V620I, and T89I represented by normalized counts. *p adj ≤ 0.1 and log 2 (fold change) ≥1 (i.e., differentially expressed).

Article Snippet: P5607; Millipore Sigma, St. Louis, MO), 10 ng/ml human transforming growth factor- β3 ( TGFβ3; cat. num.

Techniques: Expressing

Journal: eLife

Article Title: Skeletal dysplasia-causing TRPV4 mutations suppress the hypertrophic differentiation of human iPSC-derived chondrocytes

doi: 10.7554/eLife.71154

Figure Lengend Snippet: ( A ) The top 25 up-regulated genes, and their log 2 fold change, for day-56 TGFβ3-treated V620I and T89I chondrocytes compared WT. ( B ) The top 25 down-regulated genes, and their log 2 fold change, for day-56 TGFβ3-treated V620I and T89I chondrocytes compared WT.

Article Snippet: P5607; Millipore Sigma, St. Louis, MO), 10 ng/ml human transforming growth factor- β3 ( TGFβ3; cat. num.

Techniques:

Journal: eLife

Article Title: Skeletal dysplasia-causing TRPV4 mutations suppress the hypertrophic differentiation of human iPSC-derived chondrocytes

doi: 10.7554/eLife.71154

Figure Lengend Snippet: ( A ) WT chondrocytes treated with BMP4 developed a hypertrophic phenotype with enlarged lacunae, which was not present in the mutant cell lines or other conditions, as shown by Safranin-O and hematoxylin staining. Scale bar = 500 µm. Representative images from 2 experiments. ( B ) Cell diameter was significantly increased in the WT with BMP4 treatment compared to all other groups indicating a hypertrophic phenotype. Mean ± standard error of the mean (SEM). n = 249–304 cells from 2 pellets. Different letters indicate statistical significance (p < 0.05) between groups as determined by Kruskal–Wallis test with multiple comparisons since data was not normally distributed. ( C ) Western blot shows that WT had a stronger increased production of ALPL, COL10A1, IHH, RUNX2, and RUNX2-9 in response to BMP4 treatment than the mutants. ( D ) Principle component analysis (PCA) of bulk RNA-seq reveals an increased sensitivity to BMP4 (and TGFβ3 + BMP4) treatment in WT human-induced pluripotent stem cell (hiPSC)-derived chondrocytes compared to V620I and T89I. n = 3–4 samples. Figure 5—source data 1. ALPL western blot: the full raw unedited gel with and without the bands labeled. Figure 5—source data 2. COL10A1 western blot: the full raw unedited gel with and without the bands labeled. Figure 5—source data 3. IHH western blot: the full raw unedited gel with and without the bands labeled. Figure 5—source data 4. MMP13 western blot: the full raw unedited gel with and without the bands labeled. Figure 5—source data 5. RUNX2 western blot: the full raw unedited gel with and without the bands labeled. Figure 5—source data 6. GAPDH western blot: the full raw unedited gel with and without the bands labeled.

Article Snippet: P5607; Millipore Sigma, St. Louis, MO), 10 ng/ml human transforming growth factor- β3 ( TGFβ3; cat. num.

Techniques: Mutagenesis, Staining, Western Blot, RNA Sequencing Assay, Derivative Assay, Labeling

Journal: eLife

Article Title: Skeletal dysplasia-causing TRPV4 mutations suppress the hypertrophic differentiation of human iPSC-derived chondrocytes

doi: 10.7554/eLife.71154

Figure Lengend Snippet: ( A ) The full images of the ALPL, COL10A1, IHH, MMP13, RUNX2, RUNX2-9, and GAPDH western blots. ( B ) Quantification of the western blots. ( C ) Gene expression from RNA sequencing for the same genes. For simplicity, TGFβ3- and BMP4-treated groups were included in the graphs. Mean ± n = 3–4. *p < 0.05. Significance determined by Student’s t -test comparing TGFβ3- and BMP4-treated groups within the cell line and one-way analysis of variance (ANOVA) with Tukey’s post hoc test comparing cell lines within BMP4-treated group.

Article Snippet: P5607; Millipore Sigma, St. Louis, MO), 10 ng/ml human transforming growth factor- β3 ( TGFβ3; cat. num.

Techniques: Western Blot, Expressing, RNA Sequencing Assay

Journal: eLife

Article Title: Skeletal dysplasia-causing TRPV4 mutations suppress the hypertrophic differentiation of human iPSC-derived chondrocytes

doi: 10.7554/eLife.71154

Figure Lengend Snippet: ( A ) There are 9 clusters of genes based on expression and hierarchical k -means clustering of the samples. ( B ) Venn diagram shows similar and distinct differentially expressed genes (DEGs) in response to BMP4 treatment in all three lines. ( C ) Cluster 1 represented increasing in expression from TGFβ3 to BMP4 treatment (left to right on x -axis). Y -axis scale (−1.5 to 2) represents the scaled mean counts. ( D ) A protein–protein interaction network with functional enrichment analysis of cluster 1 reveals the top regulating genes and their associated concepts. Connections between protein-coding genes and Gene Ontology (GO) processes are based on the average log fold change between cell lines. Coloring of the protein-coding gene circles is divided into three to represent the log fold change for each cell line as shown in the legend. The white arrows in the legend indicates the location of the maximum log fold change for each respective cell line. The gray boxes represent the top 5 GO terms (biological process) identified for the network with the log 10 (false discovery rate) underneath the term. ( E ) A heatmap of the top 25 up-regulated genes, and their log 2 fold change, in each line compared to their respective TGFβ3 controls. ( F ) The top GO terms and biological pathways associated with the up-regulated DEGs with BMP4 treatment. Symbol color represents the cell line, and size represents the −log 10 (p adj ).

Article Snippet: P5607; Millipore Sigma, St. Louis, MO), 10 ng/ml human transforming growth factor- β3 ( TGFβ3; cat. num.

Techniques: Expressing, Functional Assay