Journal: Stem Cell Reports

Article Title: A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids

doi: 10.1016/j.stemcr.2017.11.004

Figure Lengend Snippet: Preparation of WRN CM by Lentiviral Infection (A and B) Wnt activities of recombinant hWNT3A or mWNT3A (A) and recombinant hRSPO1 or mRSPO1 (B). Activities in the absence of the hRSPO1 expression plasmid and recombinant proteins were considered as “1.” The assays were performed in three independent biological replicates. Data expressed as mean ± SEM. n.s., not significant (Student’s t test). (C–H) Mouse L cells stably expressing WRN were generated by lentiviral infection of each gene. (C–E) Wnt signaling activities of CM from L-WRN cells at densities of 2.8, 1.4, 0.7, and 0.35 × 10 6 cells/35-mm dish after 48 hr of culture (C); at a density of 1.4 × 10 6 cells/dish cultured between 24 and 96 hr (D); and at 1.4 × 10 6 cells/dish cultured for 72 hr in diluted CM or 300 ng/mL recombinant mWNT3A, with or without an hRSPO1 expression plasmid (E). Activities in the absence of WRN CM were considered as “1.” The assays were performed in three independent biological replicates. Data expressed as mean ± SEM. (F) Western blot analysis of the supernatants used in (C) undiluted (8 μL) and 4-fold diluted, and the recombinant proteins mWNT3A (1 and 0.3 ng), hRSPO1 (30 and 10 ng), and hNOG (10 and 3 ng) using anti-WNT3A, anti-RSPO1, and anti-NOG antibodies. (G) Images of human primary ileal organoids cultured in 25% WRN CM or the corresponding recombinant proteins for 7 days after a passage. Scale bars, 200 μm. (H) Wnt signaling activities of WRN CM (×1, ×1/3, ×1/10) stored in the deep freezer for 0, 6, or 12 months. The activities in the absence of CM were considered as “1.” The assay was performed in three independent biological replicates. Data expressed as mean ± SEM.

Article Snippet: When using Happy Cell ASM (Biocroi, Ireland), disrupted organoids were suspended with Happy Cell human organoid culture medium ( ) and seeded on ultra-low-attachment cell culture plate (Corning, 3473) (0.5 mL/well).

Techniques: Infection, Recombinant, Expressing, Plasmid Preparation, Stable Transfection, Generated, Cell Culture, Western Blot

Journal: Stem Cell Reports

Article Title: A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids

doi: 10.1016/j.stemcr.2017.11.004

Figure Lengend Snippet: Differentiation of TkDN4-M into Human Intestinal Organoids (A) Schematic procedures for intestinal organoid differentiation from human iPSC lines. (B) Images of the human iPSC line TkDN4-M that had been differentiated into spheroids (upper panels) and organoids (lower panels) with (right panels) or without (left panels) recombinant hWNT3A and human FGF2 (hFGF2). Arrowheads indicate spheroid- and organoid-like cell clumps. Scale bars, 200 μm. (C) Magnified images of the spheroids and organoids. Scale bars, 40 μm. (D) The numbers of spheroids per microscopic bright field in the presence or absence of WNT3A/FGF2 supplementation. The assays were performed in three independent experiments. Data are presented as mean ± SEM. ∗ p < 0.05, Student's t test. (E) Relative mRNA levels of SOX17 and CXCR4 in TkDN4-M cells differentiated into spheroids in the presence or absence of WNT3A/FGF2 supplementation were determined by qPCR and normalized to HPRT. The assay was performed in three independent biological replicates. Expression levels presented as mean ± SEM. ∗ p < 0.05, Student's t test. (F) Relative mRNA levels of the indicated genes in TkDN4-M cells during the course of differentiation (days 0, 3, and 7 [spheroids], and organoids) were determined by qPCR and normalized to HPRT . The highest expression levels of each gene in each differentiation stage were considered as “1.” The assays were performed in three independent biological replicates. Expression levels presented as mean ± SEM. (G) Images of TkDN4-M-derived organoids on day 7 after culture in 25% WRN CM, or the corresponding recombinant proteins. Scale bars, 200 μm. (H) Relative mRNA levels of LGR5 and VIL1 in TkDN4-M organoids cultured for short-term (passage 19) and long-term (passage 53) were determined by qPCR and normalized to HPRT . The assays were performed in three independent biological replicates. Expression levels presented as mean ± SEM. (I) Whole-mount images of TkDN4-M-derived organoids stained with phalloidin (blue), together with each indicated antibody. Scale bars, 50 μm.

Article Snippet: When using Happy Cell ASM (Biocroi, Ireland), disrupted organoids were suspended with Happy Cell human organoid culture medium ( ) and seeded on ultra-low-attachment cell culture plate (Corning, 3473) (0.5 mL/well).

Techniques: Recombinant, Expressing, Derivative Assay, Cell Culture, Staining

Journal: Stem Cell Reports

Article Title: A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids

doi: 10.1016/j.stemcr.2017.11.004

Figure Lengend Snippet: Comparison of Human iPSC-Derived Organoids with Primary Intestinal Organoids (A) Images of organoids from the human primary ileum or transverse colon cultured with human organoid culture medium for 6 days. Scale bars, 200 μm. (B) Relative mRNA levels of the indicated genes in organoids derived from human primary ileum, colon, and iPSCs harvested at 6 days after passage. The relative mRNA levels were determined by qPCR and normalized to GAPDH . The assays were performed in three independent biological replicates. Data are presented as mean ± SEM.

Article Snippet: When using Happy Cell ASM (Biocroi, Ireland), disrupted organoids were suspended with Happy Cell human organoid culture medium ( ) and seeded on ultra-low-attachment cell culture plate (Corning, 3473) (0.5 mL/well).

Techniques: Derivative Assay, Cell Culture

Journal: Stem Cell Reports

Article Title: A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids

doi: 10.1016/j.stemcr.2017.11.004

Figure Lengend Snippet: Lentiviral Gene Transduction into Human iPSC-Derived Intestinal Organoids (A) Schematic representation of gene transduction into organoids. TkDN4-M-derived organoids cultured in Matrigel were disrupted by a 29G needle, seeded in collagen I-coated plates, and cultured for 4 days. After 2D cultures from organoids were infected by lentiviruses, cells (including IESCs) were trypsinized, harvested, and re-embedded in Matrigel to reform organoids. (B) The proportions of Venus + organoids per microscopic bright field after 7 days of infection with IRES-Venus lentivirus. “3D” and “2D” indicate lentiviral infection into organoids harvested from Matrigel and 2D-cultured cells in collagen I-coated plates, respectively. The assay was performed in three independent biological replicates. Data are presented as mean ± SEM. ∗ p < 0.05, Student's t test. (C) Bright-field and fluorescent images of TkDN4-M-derived organoids as in (B). Scale bars, 200 μm. (D) Regenerated organoid numbers per microscopic bright field followed by 2D cultures after 7 days of infection with or without IRES-Venus lentivirus. The assay was performed in three independent biological replicates. Data are presented as mean ± SEM. (E) Bright-field and fluorescent images of TkDN4-M-derived organoids 3 weeks after infection with Wnt3a -IRES-Venus lentivirus in 2D cultures. Scale bars, 200 μm. (F) Relative mRNA levels of exogenous Wnt3a and Wnt target genes ( LGR5 , AXIN2 , and EPHB3 ) in TkDN4-M-derived organoids infected with lentiviruses as in (E). The relative mRNA levels were determined by qPCR and normalized to GAPDH . The assay was performed in three independent biological replicates. Expression levels presented as mean ± SEM. ∗ p < 0.05, Student's t test. n.d., not detected.

Article Snippet: When using Happy Cell ASM (Biocroi, Ireland), disrupted organoids were suspended with Happy Cell human organoid culture medium ( ) and seeded on ultra-low-attachment cell culture plate (Corning, 3473) (0.5 mL/well).

Techniques: Transduction, Derivative Assay, Cell Culture, Infection, Expressing

Journal: Stem Cell Reports

Article Title: A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids

doi: 10.1016/j.stemcr.2017.11.004

Figure Lengend Snippet: Growth of Human iPSC-Derived Organoids by Suspension Culture Using Happy Cell ASM (A) Time-lapse bright-field images of TkDN4-M-derived organoids cultured in Matrigel, Happy Cell ASM, or FCeM, each supplemented with 25% WRN CM and requisite ingredients for human organoid culture. Scale bars, 200 μm. (B) Whole-mount images of TkDN4-M-derived organoids cultured in Happy Cell culture medium were stained with phalloidin (blue), together with each indicated antibody. Scale bars, 50 μm. (C and D) Relative mRNA levels of the indicated genes in TkDN4-M-derived organoids cultured in Matrigel (C) or Happy Cell ASM (D) for 5 days and then exposed to each pre-stimulation medium containing the indicated concentrations of recombinant human IL-22 for 2 days. The relative mRNA levels were determined by qPCR and normalized to GAPDH . The assays were performed in three independent biological replicates. Data presented as mean ± SEM.

Article Snippet: When using Happy Cell ASM (Biocroi, Ireland), disrupted organoids were suspended with Happy Cell human organoid culture medium ( ) and seeded on ultra-low-attachment cell culture plate (Corning, 3473) (0.5 mL/well).

Techniques: Derivative Assay, Cell Culture, Staining, Recombinant

Journal: Stem Cell Reports

Article Title: A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids

doi: 10.1016/j.stemcr.2017.11.004

Figure Lengend Snippet: Characterization of Human iPSC-Derived Organoids Cultured in Happy Cell ASM (A) Relative mRNA levels of the indicated genes in TkDN4-M-derived organoids cultured with Matrigel (open bars) or Happy Cell ASM (closed bars) plus 25% WRN CM and requisite ingredients for human organoid culture after one (P1) or two (P2) passages in each culture condition. The relative mRNA levels were determined by qPCR and normalized to GAPDH . The assays were performed in three independent biological replicates. Data presented as mean ± SEM. ∗ p < 0.05, Student's t test. (B) Relative mRNA levels of the indicated genes in TkDN4-M-derived organoids cultured in Happy Cell ASM with or without 300 ng/mL recombinant mWNT3A for 6 days. The relative mRNA levels were determined by qPCR and normalized to GAPDH . The assays were performed in three independent biological replicates. Data presented as mean ± SEM. ∗ p < 0.05, Student's t test. (C) Whole-mount images of TkDN4-M-derived organoids cultured in Happy Cell ASM with or without 300 ng/mL recombinant mWNT3A after staining with phalloidin (blue) and anti-MKI67 antibodies. Scale bars, 50 μm.

Article Snippet: When using Happy Cell ASM (Biocroi, Ireland), disrupted organoids were suspended with Happy Cell human organoid culture medium ( ) and seeded on ultra-low-attachment cell culture plate (Corning, 3473) (0.5 mL/well).

Techniques: Derivative Assay, Cell Culture, Recombinant, Staining

Journal: Oncogene

Article Title: Selective inhibition of CDK9 in triple negative breast cancer

doi: 10.1038/s41388-023-02892-3

Figure Lengend Snippet: a Schematic of the two patient-derived breast organoid models (MgA1, BCMP) generated for use in this study. Representative growth curves showing ( b ) MgA1, ( c ) BCMP, and two previously described patient-derived breast cancer organoid models ( d ) HCI-010, ( e ) HCI-012, and ( f ) HCI-016 proliferation in response to escalating doses of CDDD11-8 (MgA1 F = 18.44, d.f. = 30, p < 0.0001; BCMP F = 57.65, d.f. = 30, p < 0.0001; HCI-010 F = 36.24, d.f. = 25, p < 0.0001; HCI-012 F = 20.57, d.f. = 30, p < 0.0001; HCI-016 F = 5.67, d.f. = 30, p < 0.0001). Graphed data in ( b–f ) represents the mean ± S.E.M. of 8–10 replicate wells per condition. Independent experiments were performed at least twice. Asterisks in ( b–f ) denote a statistically significant difference compared to vehicle at endpoint, as determined by Dunnett’s multiple comparisons test. f Bar graph showing the average endpoint CDDD11-8 IC 50 values for MgA1, HCI-010, HCI-016, HCI-012, and BCMP based on data presented in ( b–f ). IC 50 values were determined based on the percent of total response and fit using a four-parameter logistic function, restrained between 0 & 100%. Error bars correspond to the 95% confidence interval. h Representative immunoblot data showing dose-dependent changes in phosphorylated RNA polymerase II (p-Pol II CTD Ser2) and MCL1 protein expression in BCMP organoids after 6 h treatment with CDDD11-8. Β-Actin and total protein content are provided as loading controls. Densitometry results are presented below each set of bands, normalized to total protein content, and presented as relative to vehicle (0 nM CDDD11-8) treated cells.

Article Snippet: HCI-012 organoid culture medium is identical that used for HCI-010 and HCI-016, with the addition of 10 nM Heregulin β1 (PeproTech).

Techniques: Derivative Assay, Generated, Western Blot, Expressing