h9 human embryonic stem cells hescs  (WiCell Research Institute Inc)

Journal: Biofabrication

Article Title: Collagen hydrogel tube microbioreactors for cell and tissue manufacturing

doi: 10.1088/1758-5090/ae2718

Figure Lengend Snippet: Culturing human embryonic stem cells in ColTubes. (A) Phase-contrast images of H9 hESCs cultured in ColTubes at days 0, 1, 3, 5, and 7. (B) Live/Dead staining of H9 cells inside the tubes and released from the tubes on day 7. (C) Flow cytometry analysis shows that 99.6% of cells harvested on day 7 are Calcein AM-positive (live cells). (D) Flow cytometry analysis confirms that majority cells on day 7 express pluripotency markers OCT4 and Nanog.

Article Snippet: For a typical cell culture, H9 human embryonic stem cells (hESCs) (WA09, WiCell) loaded in 20 μ l ColTubes were suspended in 2 ml Essential 8 medium (Gibco) supplemented with 10 μ M Y-27632 (Sigma) in a 6-well plate and incubated at 37 °C with 5% CO2 and 21% O2.

Techniques: Cell Culture, Staining, Flow Cytometry

Journal: Biofabrication

Article Title: Collagen hydrogel tube microbioreactors for cell and tissue manufacturing

doi: 10.1088/1758-5090/ae2718

Figure Lengend Snippet: Differentiating H9 hESCs into Cardiomyocytes in ColTubes. (A) The cardiomyocyte production protocol. H9 hESCs are processed into ColTubes and expanded in E8 medium, followed by mesoderm induction for 1 d, cardiomyocyte differentiation from days 2–11, and metabolic enrichment from days 11–18. (B) Phase-contrast and fluorescent images of cells in ColTubes on days 0, 1, 3, 5, 7, 11, 15, and 18. Cardiomyocytes are cTnT-positive.

Article Snippet: For a typical cell culture, H9 human embryonic stem cells (hESCs) (WA09, WiCell) loaded in 20 μ l ColTubes were suspended in 2 ml Essential 8 medium (Gibco) supplemented with 10 μ M Y-27632 (Sigma) in a 6-well plate and incubated at 37 °C with 5% CO2 and 21% O2.

Techniques:

Journal: Biology Open

Article Title: Spatial heterogeneity of lactylation: insights into gene expression, metabolism, and lactate transport in human embryonic stem cells

doi: 10.1242/bio.062432

Figure Lengend Snippet: Histone Kla levels are higher in naïve-like and primed H9 hESCs than in human dermal fibroblast cells. (A) Representative phase-contrast images of naïve-like H9 hESCs (left column), primed H9 hESCs (middle column) and female hDFs (right column) treated with (lactate) and without (vehicle) 30 mM lactate for 48 h. hDF, human dermal fibroblast. Scale bars: 100 μm. (B) Representative immunoblots showing global histone acetylation (Pan Kac), global histone lactylation (Pan Kla), H3K18la, and Histone H3 in naïve-like H9 hESCs, primed H9 hESCs, male hDFs, and female hDFs treated with (+) and without (−) 30 mM lactate for 48 h before acid extraction of histones. Ponceau S was used as the loading control. (C-E) Quantified Pan Kac (C), Pan Kla (D), and H3K18la (E) protein levels. Data in C-E are mean±s.d. of three biological replicates ( N =3). Randomized block two-way ANOVA followed by an uncorrected Fisher's LSD test: * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. ns, not significant.

Article Snippet: Primed H9 hESCs (#WA09; WiCell) were maintained in mTeSRTM1 medium (#85850; STEMCELL Technologies) on Matrigel (#354277; Corning)-coated plates at atmospheric O 2 in a humidified 37°C incubator with 5% CO 2.

Techniques: Western Blot, Extraction, Control, Blocking Assay

Journal: Biology Open

Article Title: Spatial heterogeneity of lactylation: insights into gene expression, metabolism, and lactate transport in human embryonic stem cells

doi: 10.1242/bio.062432

Figure Lengend Snippet: Exogenous lactate treatment alters the expression of pluripotency genes in naïve-like and primed H9 hESCs. (A) Heatmap showing gene expression of naïve array genes in naïve-like and primed H9 hESCs treated with 30 mM lactate for 48 h relative to the vehicle, respectively. Colors on the heatmap represent fold change in gene expression of lactate-treated cells relative to the vehicle from low (blue) to high (red) expression. (B) Relative expression of naïve state-associated genes ( DNMT3L , DPPA3 , UTF1 , GBX2 , KLF4 , KLF5 , NR5A2 ) in naïve-like (top row) and primed (bottom row) H9 hESCs treated with (lactate) and without (vehicle) 30 mM lactate for 48 h . (C) Relative expression of primed state-associated genes ( DNMT3B , OTX2 , ZIC3 ) in naïve-like (top row) and primed (bottom row) H9 hESCs treated with (lactate) and without (vehicle) 30 mM lactate for 48 h. (D) Relative expression of general pluripotent state-associated genes ( LIN28A , SLC25A1 ) in naïve-like (top row) and primed (bottom row) H9 hESCs treated with (lactate) and without (vehicle) 30 mM lactate for 48 h. Data in B-D are mean±s.d. of three biological replicates ( N =3), and two to three technical replicates ( n =2-3) relative to vehicle. Ratio paired t -test or Wilcoxon matched-pairs signed rank test (primed KLF4 and primed ZIC3 ): * P <0.05, ** P <0.01, *** P <0.001. ns, not significant.

Article Snippet: Primed H9 hESCs (#WA09; WiCell) were maintained in mTeSRTM1 medium (#85850; STEMCELL Technologies) on Matrigel (#354277; Corning)-coated plates at atmospheric O 2 in a humidified 37°C incubator with 5% CO 2.

Techniques: Expressing, Gene Expression

Journal: Biology Open

Article Title: Spatial heterogeneity of lactylation: insights into gene expression, metabolism, and lactate transport in human embryonic stem cells

doi: 10.1242/bio.062432

Figure Lengend Snippet: LDHA levels are higher centrally, regardless of treatment, while OCT4 becomes more centrally localized with oxamate treatment in naïve-like hESCs. (A) Representative immunofluorescence images of naïve-like H9 hESC colonies under vehicle conditions, treated with 30 mM lactate, 10 mM oxamate, 250 nM AZD, 100 μM CBX for 48 h stained with DAPI (blue), LDHA (green) and OCT4 (red). Bottom row are representative immunofluorescence images of no primary control condition. Scale bars: 50 μm. (B,C) Quantified mean fluorescence intensity of LDHA (B) and OCT4 (C) in vehicle-, lactate-, oxamate-, AZD-, and CBX-treated naïve-like H9 hESCs. Statistical analyses in B and C were performed using mean±s.d. of three biological replicates ( N =3) relative to no treatment control (NTC) and nine to 15 technical replicates ( n =9-15). Graphs shown in B and C display technical replicates that are not standardized to NTC. Ordinary one-way ANOVA followed by Dunnett's multiple comparisons test or Kruskal–Wallis test followed by Dunn's multiple comparisons test (OCT4 total colony). (D,E) Quantified mean fluorescence intensity of LDHA (D) and OCT4 (E) in the vehicle, lactate-, oxamate-, AZD-, and CBX-treated naïve-like H9 HESC outer, middle, and inner colony sections relative to the total colony mean fluorescence intensity. Statistical analyses in D and E were performed using mean±s.d. of three biological replicates ( N =3) and nine to 15 technical replicates ( n =9-15). Graphs in D and E display technical replicates that are not standardized to the no-treatment control. Ordinary one-way ANOVA followed by Tukey's multiple comparisons test or Kruskal–Wallis test followed by Dunn's multiple comparisons test (LDHA vehicle, LDHA lactate): * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Primed H9 hESCs (#WA09; WiCell) were maintained in mTeSRTM1 medium (#85850; STEMCELL Technologies) on Matrigel (#354277; Corning)-coated plates at atmospheric O 2 in a humidified 37°C incubator with 5% CO 2.

Techniques: Immunofluorescence, Staining, Control, Fluorescence