Journal: Cancer letters

Article Title: NCAM- and FGF-2-mediated FGFR1 signaling in the tumor microenvironment of esophageal cancer regulates the survival and migration of tumor-associated macrophages and cancer cells.

doi: 10.1016/j.canlet.2016.06.009

Figure Lengend Snippet: Fig. 1. Induction of NCAM in peripheral blood monocyte (PBMo)-derived macrophages stimulated with conditioned medium of a TE series ESCC cell line (TECM). PBMos were treated with 25 ng/mL recombinant human M-CSF for 6 d to induce Macrophage_L and then exposed to the TECM (TE-8, TE-9 and TE-15) for 2 d to induce TAM_Ls. (A) NCAM mRNA induction in the TAM_Ls was confirmed by RT-PCR (left panel). The results were normalized to GAPDH as a control, and densitometric analysis of bands was performed with ImageJ (National Institutes of Health, Maryland, USA) (right panel). The results are the mean ± SEM (n = 3, *p < 0.05). (B) NCAM expression levels in the TAM_Ls were determined by quantitative RT-PCR, compared with those of the Macrophage_Ls, and normalized to GAPDH expression. The results are the mean ± SEM (n = 3, *p < 0.05). (C) NCAM induction was also confirmed in the TAM_Ls by western blotting (left panel). The results were normalized to β-actin as a control, and densitometric analysis of bands was performed with the ImageJ (right panel). The results are the mean ± SEM (n = 3, *p < 0.05). (D) NCAM and CD11b expression in Macrophage_Ls and TAM_Ls. Double immunofluorescence was performed using anti-NCAM (green) plus the macrophage marker anti-CD11b (red). NCAM expression was detectable in TAM_Ls_TE-9 (left panel). Nuclei were stained with DAPI (blue). Scale bar, 10 μm. NCAM fluorescent intensity data were quantified using ImageJ (right panel). The corrected total cell flu- orescence (CTCE) was calculated as integrated density −(area of selected cell × mean fluorescence of background reading). The results are the mean ± SEM (n = 3, **p < 0.001). (E) The localization of NCAM and F-actin in TAM_Ls_TE-9. Immunofluorescence was performed using anti-NCAM (green) in TAM_Ls_TE-9. Nuclei and F-actin were stained with DAPI (blue) and phalloidin (red), respectively. NCAM and F-actin were co-localized at a part of the lamellipodia (arrowheads). Scale bar, 10 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The following selective inhibitors and recombinant protein were used: PI3K inhibitor (LY294002, 20 μM; Cell Signaling Technology, Beverly, MA); FGFR1 inhibitor (SU5402, 50 μM; Santa Cruz Biotechnology, Dallas, TX); human recombinant basic fibroblast growth factor (rhFGF-2, 5–20 ng/mL; R&D Systems).

Techniques: Derivative Assay, Recombinant, Reverse Transcription Polymerase Chain Reaction, Control, Expressing, Quantitative RT-PCR, Western Blot, Marker, Staining

Journal: Biomaterials

Article Title: Sustained release and protein stabilization reduce the growth factor dosage required for human pluripotent stem cell expansion.

doi: 10.1016/j.biomaterials.2020.120007

Figure Lengend Snippet: Fig. 1. MCMs bind and release bFGF for sustained bFGF presentation in hPSC culture. (A) Schematic for binding of bFGF to mineral-coated microparticles (MCMs) in solution. (B) Proposed model for presentation and presence of active bFGF over time in (top) conventional chemically-defined hPSC culture or (bottom) culture with bFGF-loaded MCMs (bFGF-MCMs). bFGF loses activity over time when delivered as soluble protein via daily media changes in culture, while MCMs stabilize and allow sustained release of active bFGF. (C) Schematic representation of two culture formats employed in this study: Transwell culture, in which MCMs release bFGF from Transwells, and direct culture, in which MCMs releasing bFGF are added directly to cells. (D) Photographs and scanning electron micrographs of bFGF-MCMs in (top) 12-well Transwell format and (bottom) direct culture format with hPSCs. Scale bar = 10 μm.

Article Snippet: For MCM loading, sterilized MCMs were suspended in sterile PBS to make a 1 mg/mL stock solution, and appropriate volumes of recombinant human basic fibroblast growth factor (bFGF, carrier-free; R& D Systems) and MCM stock solutions were combined to achieve the desired final concentration of each in the loading solution (e.g., 1.0 μg/ mL bFGF, 1.0 mg/mL MCMs for non-optimized loading; 0.456 μg/mL bFGF, 0.375 mg/mL MCMs for optimized loading).

Techniques: Binding Assay, Activity Assay

Journal: Biomaterials

Article Title: Sustained release and protein stabilization reduce the growth factor dosage required for human pluripotent stem cell expansion.

doi: 10.1016/j.biomaterials.2020.120007

Figure Lengend Snippet: Fig. 2. bFGF-loaded MCMs (bFGF-MCMs) in Transwell culture have a dose-dependent effect on the percentage of hPSCs expressing Oct4 and Nanog. (A) Representative images of H1 hPSCs at passage 3 (day 12) of culture in E8 (control) or E7 with varying doses of bFGF-MCMs (scale bar = 250 μm). Colonies with normal stem cell morphology were observed in the E8 control and E7 + high bFGF-MCM conditions, while varying degrees of spontaneous differentiation were seen in the E7, E7 + low bFGF-MCM, and E7 + med bFGF-MCM conditions (white arrows). (B) Quantification of Oct4/Nanog expression in hPSCs grown with or without bFGF-MCMs in Transwell culture for 3 passages (n = 3, error bars = s.d.), as assessed by flow cytometry. n = 3, error bars = s.d. bFGF utilization as denoted on the right y-axis was calculated based on amount of bFGF used to maintain a single well of hPSCs in a 12-well plate format (1 mL media/well) for three passages, relative to E8 control (100%). (C) Representative flow cytometry plots of hPSCs after Transwell culture with E7 containing low, medium, or high doses of bFGF-MCMs for 3 passages.

Article Snippet: For MCM loading, sterilized MCMs were suspended in sterile PBS to make a 1 mg/mL stock solution, and appropriate volumes of recombinant human basic fibroblast growth factor (bFGF, carrier-free; R& D Systems) and MCM stock solutions were combined to achieve the desired final concentration of each in the loading solution (e.g., 1.0 μg/ mL bFGF, 1.0 mg/mL MCMs for non-optimized loading; 0.456 μg/mL bFGF, 0.375 mg/mL MCMs for optimized loading).

Techniques: Expressing, Control, Cytometry

Journal: Biomaterials

Article Title: Sustained release and protein stabilization reduce the growth factor dosage required for human pluripotent stem cell expansion.

doi: 10.1016/j.biomaterials.2020.120007

Figure Lengend Snippet: Fig. 3. Design of experiments (DOE) optimization of bFGF binding solution identifies MCM conditions that maintain hPSC pluripotency marker expression while minimizing bFGF usage. (A) Representation of a five-level DOE experimental space. The levels were centered around the point (0,0) of 1 mg/mL of MCMs 1 μg/mL bFGF, with 2-fold based steps above (+) and below (−), and 2.67-fold axial points above (A) and below (a) the center point. The concentrations of bFGF and MCMs in the binding solution were varied while the total amount of bFGF-MCMs used in Transwell culture was held constant. %Oct4+/Nanog+ hPSCs at passage 3 was measured as the response variable. (B) Flow cytometry quantification of %Oct4+/Nanog+ hPSCs cultured with bFGF-MCMs from each DOE condition for 3 passages. n = 3, error bars = s.d. (C) Representative images of hPSCs in E8 medium, E7 medium, and the a0 DOE condition in E7. (D) Results of the DOE-generated model. The model allowed for identification of an optimized binding solution that minimizes bFGF utilization with a predicted 95% Oct4+/Nanog+ cell population at passage 3 of Transwell culture. (E) Comparison of the performance of DOE-optimized MCMs vs. non-optimized MCMs in maintaining hPSC pluripotency in Transwell (left) and direct (right) culture formats. For each culture format, four bFGF-MCM doses were tested, with total bFGF utilization matched between optimized and non-optimized MCMs for each respective dose. bFGF utilization as denoted on the x-axis was calculated based on amount of bFGF used to maintain a single well of hPSCs in a 12-well plate format (1 mL media/well) for three passages (see Supplementary Table S2). n = 3, error bars = s.d.; ****p < 0.0001, **p < 0.01; two-way ANOVA. “n.d.” denotes a condition for which P3 flow cytometry data were not collected due to inability of the hPSCs to be effectively passaged at the corresponding dose of non- optimized bFGF-MCMs.

Article Snippet: For MCM loading, sterilized MCMs were suspended in sterile PBS to make a 1 mg/mL stock solution, and appropriate volumes of recombinant human basic fibroblast growth factor (bFGF, carrier-free; R& D Systems) and MCM stock solutions were combined to achieve the desired final concentration of each in the loading solution (e.g., 1.0 μg/ mL bFGF, 1.0 mg/mL MCMs for non-optimized loading; 0.456 μg/mL bFGF, 0.375 mg/mL MCMs for optimized loading).

Techniques: Binding Assay, Marker, Expressing, Flow Cytometry, Cell Culture, Generated, Comparison, Cytometry

Journal: Biomaterials

Article Title: Sustained release and protein stabilization reduce the growth factor dosage required for human pluripotent stem cell expansion.

doi: 10.1016/j.biomaterials.2020.120007

Figure Lengend Snippet: Fig. 4. Direct culture with optimized bFGF-MCMs maintains hPSC pluripotency and normal karyotype during long-term culture. (A) Comparison of colony mor- phology in H1 hESCs at passages 3 and 25 with bFGF-MCMs. (B) hPSCs in direct culture with bFGF-MCMs can be transitioned back to E8/Matrigel with minimal MCM carryover within 2 passages, and display normal hPSC colony morphology. Scale bars = 100 μm. (C) hPSCs in direct culture with bFGF-MCMs for 25 passages maintain robust expression of pluripotency markers Oct4 and Nanog. Scale bar = 100 μm. (D) G-banded karyotyping of hPSCs maintained in direct culture with optimized bFGF-MCMs for 25 passages. (E) hPSCs in direct culture with bFGF-MCMs retain the potential to spontaneously differentiate into derivatives of the three primary germ layers. EBs were formed, allowed to spontaneously differentiate and adhere to Matrigel-coated dishes, and stained for markers of (i) ectoderm (beta-III tubulin), (ii) mesoderm (alpha smooth muscle actin), and (iii) endoderm (alpha-fetoprotein) lineages. Scale bars = 50 μm. (F) Histological analysis of teratomas generated from hPSCs after long-term (25 passages) direct culture with bFGF-MCMs. Differentiation into all three germ layers is shown: (i) ectoderm (neuroe- pithelium, pigmented retinal tissue), (ii) mesoderm (cartilage) and (iii) endoderm (liver). Images shown in (E) and (F) are for H1 hESCs. Scale bar = 50 μm.

Article Snippet: For MCM loading, sterilized MCMs were suspended in sterile PBS to make a 1 mg/mL stock solution, and appropriate volumes of recombinant human basic fibroblast growth factor (bFGF, carrier-free; R& D Systems) and MCM stock solutions were combined to achieve the desired final concentration of each in the loading solution (e.g., 1.0 μg/ mL bFGF, 1.0 mg/mL MCMs for non-optimized loading; 0.456 μg/mL bFGF, 0.375 mg/mL MCMs for optimized loading).

Techniques: Comparison, Expressing, Staining, Generated

Journal: Biomaterials

Article Title: Sustained release and protein stabilization reduce the growth factor dosage required for human pluripotent stem cell expansion.

doi: 10.1016/j.biomaterials.2020.120007

Figure Lengend Snippet: Fig. 5. Local delivery increases growth factor biological potency. (A) %Oct4/Nanog expression of hPSCs cultured with the same amount of optimized bFGF-MCMs in either Transwell or direct culture. In this experiment, fresh MCMs were replaced at each passage (i.e., every 4 days) in both Transwell and direct culture formats. n = 3, error bars = s.d.; ****p < 0.0001, two-way ANOVA. (B) Local delivery (i.e., direct culture with bFGF-MCMs). amplifies growth factor activity at the culture surface, as measured in a cell-free bFGF bioactivity assay using the Quantikine bFGF ELISA kit. n = 3, error bars = s.d.; *p < 0.05, t-test.

Article Snippet: For MCM loading, sterilized MCMs were suspended in sterile PBS to make a 1 mg/mL stock solution, and appropriate volumes of recombinant human basic fibroblast growth factor (bFGF, carrier-free; R& D Systems) and MCM stock solutions were combined to achieve the desired final concentration of each in the loading solution (e.g., 1.0 μg/ mL bFGF, 1.0 mg/mL MCMs for non-optimized loading; 0.456 μg/mL bFGF, 0.375 mg/mL MCMs for optimized loading).

Techniques: Expressing, Cell Culture, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Biomaterials

Article Title: Sustained release and protein stabilization reduce the growth factor dosage required for human pluripotent stem cell expansion.

doi: 10.1016/j.biomaterials.2020.120007

Figure Lengend Snippet: Fig. 6. Binding to MCMs improves bFGF thermal stability. (A) bFGF bound to and released from MCMs maintains hPSC pluripotency marker expression more effectively than a matched amount of soluble bFGF (“Bolus”). “4D” denotes media changes every 4 days (i.e., only at the time of passaging). (B) bFGF bound to MCMs is stabilized against activity loss during incubation at physiological temperatures, as measured by Quantikine bFGF ELISA. “%activity remaining” is expressed relative to 4°C storage of each respective condition. (C) Comparison of total bFGF protein release from bFGF-MCMs vs. PLGA microspheres at 37°C. (D) Comparison of active bFGF protein release from bFGF-MCMs vs. PLGA microspheres. Values for cumulative daily release were extrapolated based on 2 h release in the Quantikine bFGF ELISA for each time point assessed. Asterisks indicate statistically significant difference compared to (A) E8 control, (B) E7+soluble bFGF, (C–D) PLGA microspheres. n = 3, error bars = s.d.; p < 0.05 (*), 0.01 (**), 0.001 (**), or 0.0001 (****), two-way ANOVA.

Article Snippet: For MCM loading, sterilized MCMs were suspended in sterile PBS to make a 1 mg/mL stock solution, and appropriate volumes of recombinant human basic fibroblast growth factor (bFGF, carrier-free; R& D Systems) and MCM stock solutions were combined to achieve the desired final concentration of each in the loading solution (e.g., 1.0 μg/ mL bFGF, 1.0 mg/mL MCMs for non-optimized loading; 0.456 μg/mL bFGF, 0.375 mg/mL MCMs for optimized loading).

Techniques: Binding Assay, Marker, Expressing, Passaging, Activity Assay, Incubation, Enzyme-linked Immunosorbent Assay, Comparison, Control