Journal: Stem Cell Research & Therapy

Article Title: Protease inhibitors enhance extracellular collagen fibril deposition in human mesenchymal stem cells

doi: 10.1186/s13287-015-0191-1

Figure Lengend Snippet: Collagen internalization and degradation by hMSCs (P6). a - f Two-dimensional monolayer culture: a - c 1-hour incubation in serum-free medium ( green : Alexa Fluor 488 labeled collagen; 25 μg/ml; blue : nucleus labeled by DAPI); d - f 24-hour incubation in serum-free medium where DQ FITC-labeled collagen was 10 % of total collagen content (20 μg/ml) ( green : fluorescein-conjugated DQ-collagen type I; red : lysosomes). g - i Three-dimensional culture in collagen microspheres at different time points: g 2 mg/ml type I rat collagen with cell density at 1 × 10 5 per ml at 3 h after encapsulation; h 1 mg/ml type I rat collagen with cell density at 5 × 10 5 per ml at 19 h after encapsulation; i 1 mg/ml type I rat collagen with cell density at 5 × 10 5 per ml at 23 h after encapsulation; i1 and i2 Side views of hMSCs inside a collagen microsphere ( green : 1 % fluorescein-labeled bovine collagen; red : LysoTracker-labeled cell). DAPI 4ʹ,6-diamidino-2-phenylindole, FITC fluorescein isothiocyanate, hMSC human mesenchymal stem cell

Article Snippet: Samples were blocked with 2 % normal horse serum for non-specific binding and then incubated with a primary antibody against human type I collagen (rat anti-human type I collagen antibody in 1:200 dilution; Chondrex, Redmond, WA, USA) overnight at 4 °C.

Techniques: Incubation, Labeling

Journal: Stem Cell Research & Therapy

Article Title: Protease inhibitors enhance extracellular collagen fibril deposition in human mesenchymal stem cells

doi: 10.1186/s13287-015-0191-1

Figure Lengend Snippet: Extracellular collagen fibril formation by hMSCs in different treatment groups at different time points. a - d 4 h. e - h 24 h. i - l 48 h. m - p 72 h. q - t Day 11. a , e , i , m , q Control (normal medium). b , f , j , n , r Inhibition of intracellular matrix degradation by intracellular cysteine proteinase inhibitor E64D. c , g , k , o , s Inhibition of extracellular matrix degradation by wide range collagenase inhibitor GM6001. d , h , l , p , t Inhibition of both intracellular and extracellular matrix degradation. E64D (20 μM); GM6001 (25 μM); green : Alexa Fluor 488-labeled collagen type I at 10 μg/ml; orange : Lysosomes. u Quantitative analysis of percentage of collagen content in extracellular fibril fraction under different treatments and at different time points (n = 2 with duplicates). hMSC human mesenchymal stem cell

Article Snippet: Samples were blocked with 2 % normal horse serum for non-specific binding and then incubated with a primary antibody against human type I collagen (rat anti-human type I collagen antibody in 1:200 dilution; Chondrex, Redmond, WA, USA) overnight at 4 °C.

Techniques: Inhibition, Labeling

Journal: Stem Cell Research & Therapy

Article Title: Protease inhibitors enhance extracellular collagen fibril deposition in human mesenchymal stem cells

doi: 10.1186/s13287-015-0191-1

Figure Lengend Snippet: Intracellular collagen fibril formation and growth by hMSCs in different groups at different time points after removal of extracellular collagen fibrils by treatment with bacterial collagenase type VI. a - p Visualization of internalized collagen after collagenase treatment. a - d 2 h. e - h , m , n 4 h. i - l 6 h. o , p 24 h. a , e , i Control. b , f , j Inhibition of intracellular matrix degradation by cysteinase inhibitor (E64D). c , g , k , m Combination of both intracellular (E64D) and extracellular (GM6001) protease inhibitors. d , h , l , n - p Inhibition of extracellular matrix degradation by broad-spectrum protease inhibitor (GM6001). m 1 – 2, o1-2 Side views of hMSCs showing that the location of the stained fibrils was within the intracellular space. q - t Quantitative measurement of total fluorescence intensity of internalized collagen per cell on images at 63× (mean + standard deviation, n = 2 to 8). q Control. r Inhibition of intracellular matrix degradation by cysteine proteinase inhibitor (E64D). s Combination of both intracellular (E64D) and extracellular (GM6001) protease inhibitors. t Inhibition of extracellular matrix degradation by broad-spectrum protease inhibitor (GM6001). Green : Alexa Fluor 488-labeled collagen type I; orange : lysozyme; red : Plasma membrane; and grey : reflection. Collagen (10 mg/ml), E64D (20 μM), and GM6001 (25 μM) were used. hMSC human mesenchymal stem cell

Article Snippet: Samples were blocked with 2 % normal horse serum for non-specific binding and then incubated with a primary antibody against human type I collagen (rat anti-human type I collagen antibody in 1:200 dilution; Chondrex, Redmond, WA, USA) overnight at 4 °C.

Techniques: Inhibition, Protease Inhibitor, Staining, Fluorescence, Standard Deviation, Labeling

Journal: Stem Cell Research & Therapy

Article Title: Protease inhibitors enhance extracellular collagen fibril deposition in human mesenchymal stem cells

doi: 10.1186/s13287-015-0191-1

Figure Lengend Snippet: Collagen fibril deposition by hMSCs with and without exogenously supplemented collagen or ascorbic acid or both. a - d Immunofluorescence staining of exogenous type I collagen (rat) and newly synthesized type I collagen (human) in hMSCs without ascorbic acid but with exogenously supplemented collagen: a Merged channels. b Rat type I collagen. c Human type I collagen. d Nuclei. e - h Immunofluorescence staining of collagen type I in hMSCs with ascorbic acid but without exogenously supplemented collagen. i - l Immunofluorescence staining of human type I collagen and fluorescence-labeled exogenous rat type I collagen when vitamin C and exogenous collagen were co-supplemented. e , i Control. f , j Intracellular protease inhibitor E64D. g , k Combination of intracellular (E64D) and extracellular (GM6001) protease inhibitors. h , l Extracellular protease inhibitor GM6001. For ( e - l ), green : human type I collagen; red : Exogenously supplemented fluorescence-labeled rat tail collagen type I; blue : 4ʹ,6-diamidino-2-phenylindole (DAPI). hMSC human mesenchymal stem cell

Article Snippet: Samples were blocked with 2 % normal horse serum for non-specific binding and then incubated with a primary antibody against human type I collagen (rat anti-human type I collagen antibody in 1:200 dilution; Chondrex, Redmond, WA, USA) overnight at 4 °C.

Techniques: Immunofluorescence, Staining, Synthesized, Fluorescence, Labeling, Protease Inhibitor