Figure 5
BM-MSCs from Sparc−/− mice are defective in collagen type I deposition despite high TGF-β1 synthesis. (A) In vitro confocal microscopic analysis for collagen type I (green) and SPARC (red) expression in BM-MSCs obtained from WT and Sparc−/− mice. BM-MSCs were seeded onto culture dishes adapted for confocal microscopy, starved for 24 hours, and then treated with recombinant TGF-β1. Untreated cells served as the control. The images show that rTGF-β1 administration induced SPARC up-regulation and collagen type I fiber deposition only in WT BM-MSCs. Results of 1 representative experiment of the 3 performed in triplicate are shown. Original magnifications ×400. Scale bars represent 50 μm. (B) Western blot analysis for collagen type I, SPARC, and TGF-β1 on whole cell lysates from BM-MSCs treated rTGF-β1. Untreated cells served as the control. Western blot analysis highlights a different pattern of collagen type I production by WT and Sparc−/− BM-MSCs. Specifically, WT BM-MSCs produce both the mature form of collagen (79 kDa) and precursors of low molecular weight (predicted molecular weight of 107-120 kDa). In contrast, Sparc−/− BM-MSCs were unable to produce the mature form of collagen (79 kDa) and accumulated precursors of high molecular weight (223 kDa), which indicates a defect in collagen maturation in Sparc−/−, but not WT, BM-MSCs. WB analysis also showed that WT BM-MSCs up-regulate SPARC expression after rTGF-β1 administration. The results of 1 representative experiments of the 3 performed in triplicate are shown. (C) Western blot quantitative analysis of SPARC expression performed on WT BM-MSCs treated with rTGF-β1. Untreated cells served as the control. The data represent 1 experiment of the 3 performed in triplicate. (D) Immunohistochemical analysis of collagen type I expression (DAB, brown signal) in BM samples from mouse chimeras showing that chimeric mice with WT stroma (WT > WT, Sparc−/− > WT) have significantly higher interstitial deposition of collagen type I (left panels, black arrows) compared with chimeras with Sparc−/− BM stroma (WT > Sparc−/−, Sparc−/− > Sparc−/−; right panels). Four representative immunostained sections (1 per group) are shown of the 20 evaluated. Original magnifications ×400. Scale bars represent 50 μm. (E) Immunohistochemical analysis of TGF-β1 expression (DAB, brown signal) in BM samples from TPO-treated mouse chimeras showing that a higher density of BM stromal cells expressing TGF-β1 (arrows) is detected in chimeras with a Sparc−/− BM stroma (WT > Sparc−/−, Sparc−/− > Sparc−/−; right panels) compared with the WT counterpart (WT > WT, Sparc−/− > WT; left panels). Four representative immunostained sections (1 per group) are shown of the 20 evaluated. Original magnifications ×400. Scale bars represent 50 μm.

BM-MSCs from Sparc−/− mice are defective in collagen type I deposition despite high TGF-β1 synthesis. (A) In vitro confocal microscopic analysis for collagen type I (green) and SPARC (red) expression in BM-MSCs obtained from WT and Sparc−/− mice. BM-MSCs were seeded onto culture dishes adapted for confocal microscopy, starved for 24 hours, and then treated with recombinant TGF-β1. Untreated cells served as the control. The images show that rTGF-β1 administration induced SPARC up-regulation and collagen type I fiber deposition only in WT BM-MSCs. Results of 1 representative experiment of the 3 performed in triplicate are shown. Original magnifications ×400. Scale bars represent 50 μm. (B) Western blot analysis for collagen type I, SPARC, and TGF-β1 on whole cell lysates from BM-MSCs treated rTGF-β1. Untreated cells served as the control. Western blot analysis highlights a different pattern of collagen type I production by WT and Sparc−/− BM-MSCs. Specifically, WT BM-MSCs produce both the mature form of collagen (79 kDa) and precursors of low molecular weight (predicted molecular weight of 107-120 kDa). In contrast, Sparc−/− BM-MSCs were unable to produce the mature form of collagen (79 kDa) and accumulated precursors of high molecular weight (223 kDa), which indicates a defect in collagen maturation in Sparc−/−, but not WT, BM-MSCs. WB analysis also showed that WT BM-MSCs up-regulate SPARC expression after rTGF-β1 administration. The results of 1 representative experiments of the 3 performed in triplicate are shown. (C) Western blot quantitative analysis of SPARC expression performed on WT BM-MSCs treated with rTGF-β1. Untreated cells served as the control. The data represent 1 experiment of the 3 performed in triplicate. (D) Immunohistochemical analysis of collagen type I expression (DAB, brown signal) in BM samples from mouse chimeras showing that chimeric mice with WT stroma (WT > WT, Sparc−/− > WT) have significantly higher interstitial deposition of collagen type I (left panels, black arrows) compared with chimeras with Sparc−/− BM stroma (WT > Sparc−/−, Sparc−/− > Sparc−/−; right panels). Four representative immunostained sections (1 per group) are shown of the 20 evaluated. Original magnifications ×400. Scale bars represent 50 μm. (E) Immunohistochemical analysis of TGF-β1 expression (DAB, brown signal) in BM samples from TPO-treated mouse chimeras showing that a higher density of BM stromal cells expressing TGF-β1 (arrows) is detected in chimeras with a Sparc−/− BM stroma (WT > Sparc−/−, Sparc−/− > Sparc−/−; right panels) compared with the WT counterpart (WT > WT, Sparc−/− > WT; left panels). Four representative immunostained sections (1 per group) are shown of the 20 evaluated. Original magnifications ×400. Scale bars represent 50 μm.

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