Journal: Biology

Article Title: Thermostable Human Basic Fibroblast Growth Factor (TS-bFGF) Engineered with a Disulfide Bond Demonstrates Superior Culture Outcomes in Human Pluripotent Stem Cell

doi: 10.3390/biology12060888

Figure Lengend Snippet: Computational design of thermostable-bFGF (TS-bFGF) and its comparison with wild-type bFGF (WT-bFGF) in terms of stability. ( A ) Molecular images of WT-bFGF and TS-bFGF carrying an engineered disulfide (S-S) bond. The dotted green circles indicate the regions engineered in detail. ( B ) Docking images of WT-bFGF and TS-bFGF with the FGF receptor. ( C ) Thermal stability (Tm, melting temperature) of WT-bFGF and TS-bFGF. ( D ) Cell proliferation activity of WT-bFGF and TS-bFGF in NIH/3T3 cells stored in solution at 25 °C for a period of 12 months. The values at each point were normalized by setting the values of control (a fresh WT-bFGF of TS-bFGF, not stored at 25 °C) as 100%. *** p < 0.001.

Article Snippet: After three days, the medium was completely replaced with a hESC proliferation medium containing WT-bFGF (Peprotech, USA) or TS-bFGF with 10 µM Y-27632 (MedChemExpress, USA).

Techniques: Activity Assay

Journal: Biology

Article Title: Thermostable Human Basic Fibroblast Growth Factor (TS-bFGF) Engineered with a Disulfide Bond Demonstrates Superior Culture Outcomes in Human Pluripotent Stem Cell

doi: 10.3390/biology12060888

Figure Lengend Snippet: Comparison of luciferase activity under culture conditions in 293T cells. ( A ) Schematic diagram of the luciferase reporter gene constructs and scheme (SRE with luc2p and serum-response element with luciferase gene, respectively. ( B ) Luciferase activity of reporters carrying either WT-bFGF or TS-bFGF were measured in hESC. ( C ) Luciferase activity of reporters carrying either the heat-incubated WT-bFGF or TS-bFGF were measured in hESCs at 12 and 24 h. Values are expressed as the mean ± SEM of three independent experiments. *** p < 0.001.

Article Snippet: After three days, the medium was completely replaced with a hESC proliferation medium containing WT-bFGF (Peprotech, USA) or TS-bFGF with 10 µM Y-27632 (MedChemExpress, USA).

Techniques: Luciferase, Activity Assay, Construct, Incubation

Journal: Biology

Article Title: Thermostable Human Basic Fibroblast Growth Factor (TS-bFGF) Engineered with a Disulfide Bond Demonstrates Superior Culture Outcomes in Human Pluripotent Stem Cell

doi: 10.3390/biology12060888

Figure Lengend Snippet: Analysis of the comparative effect of hESC proliferation and stemness using WT-bFGF and TS-bFGF. ( A ) Phase contrast images of hESC colonies grown under different bFGF conditions on feeder-free layers on day 2. ( B ) Comparative analysis of growth rates of hESCs treated with WT-bFGF and TS-bFGF in continuous cell culture. ( C ) Comparison of cumulative population cell number over time in WT-bFGF-cultured and TS-bFGF-cultured hESCs in short-term (passage 5) or long-term (passage 10) cell culture. ( D ) Relative quantification of OCT4, SOX2, and KLF4 mRNA levels of the hESC colonies on day 3 of culture with WT-bFGF or TS-bFGF. Scale bar: 200 µm. Values are expressed as the mean ± SEM of three independent experiments. n.s: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After three days, the medium was completely replaced with a hESC proliferation medium containing WT-bFGF (Peprotech, USA) or TS-bFGF with 10 µM Y-27632 (MedChemExpress, USA).

Techniques: Cell Culture

Journal: Biology

Article Title: Thermostable Human Basic Fibroblast Growth Factor (TS-bFGF) Engineered with a Disulfide Bond Demonstrates Superior Culture Outcomes in Human Pluripotent Stem Cell

doi: 10.3390/biology12060888

Figure Lengend Snippet: Analysis of main characteristics of hESCs cultured with TS-bFGF. ( A ) Effects of WT-bFGF and TS-bFGF on hESC cycle. ( B ) hESC colonies were positively stained with alkaline phosphatase. Immunofluorescence staining was performed to detect pluripotent markers (Scale bar: 200 μm). ( C , D ) Representative immunofluorescence images showing the expression of pluripotency markers (SSEA4 and TRA-1-60) in hESCs cultured with either TS-bFGF or WT-bFGF. Nuclei were stained with DAPI (Scale bar: 50 μm). Values are expressed as the mean ± SEM of three independent experiments. n.s: not significant, * p < 0.05, ** p < 0.01.

Article Snippet: After three days, the medium was completely replaced with a hESC proliferation medium containing WT-bFGF (Peprotech, USA) or TS-bFGF with 10 µM Y-27632 (MedChemExpress, USA).

Techniques: Cell Culture, Staining, Immunofluorescence, Expressing

Journal: Biology

Article Title: Thermostable Human Basic Fibroblast Growth Factor (TS-bFGF) Engineered with a Disulfide Bond Demonstrates Superior Culture Outcomes in Human Pluripotent Stem Cell

doi: 10.3390/biology12060888

Figure Lengend Snippet: Three-lineage differentiation efficiency of hESCs as indicated by gene expression. ( A ) Schematic of the EB formation procedure. One day before initiation of differentiation, WT-bFGF or TS-bFGF was added to the hESCs, which were then transferred into AggreWell plates at 24 h. The EBs formed through spontaneous differentiation were transferred into untreated 6-well plates without bFGF. Comparative evaluation of enhanced differentiation capacity into mesoderm ( B ), ectoderm ( C ), and endoderm ( D ) of WT-bFGF or TS-bFGF-treated hESC-derived EBs. Values are expressed as the mean ± SEM of three independent experiments. n.s: not significant, * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: After three days, the medium was completely replaced with a hESC proliferation medium containing WT-bFGF (Peprotech, USA) or TS-bFGF with 10 µM Y-27632 (MedChemExpress, USA).

Techniques: Expressing, Derivative Assay