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. 2016 Apr;157(4):1348-56.
doi: 10.1210/en.2015-1986. Epub 2016 Feb 12.

Transient Suppression of TGFβ Receptor Signaling Facilitates Human Islet Transplantation

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Free PMC article

Transient Suppression of TGFβ Receptor Signaling Facilitates Human Islet Transplantation

Xiangwei Xiao et al. Endocrinology. .
Free PMC article

Abstract

Although islet transplantation is an effective treatment for severe diabetes, its broad application is greatly limited due to a shortage of donor islets. Suppression of TGFβ receptor signaling in β-cells has been shown to increase β-cell proliferation in mice, but has not been rigorously examined in humans. Here, treatment of human islets with a TGFβ receptor I inhibitor, SB-431542 (SB), significantly improved C-peptide secretion by β-cells, and significantly increased β-cell number by increasing β-cell proliferation. In addition, SB increased cell-cycle activators and decreased cell-cycle suppressors in human β-cells. Transplantation of SB-treated human islets into diabetic immune-deficient mice resulted in significant improvement in blood glucose control, significantly higher serum and graft insulin content, and significantly greater increases in β-cell proliferation in the graft, compared with controls. Thus, our data suggest that transient suppression of TGFβ receptor signaling may improve the outcome of human islet transplantation, seemingly through increasing β-cell number and function.

Figures

Figure 1.
Figure 1.
TGFbRII is exclusively detected in β-cells in human islets. A and B, Representative immunostaining images for TGFbRI (red), insulin (green), and HO (blue) (A) and TGFbRI (red), glucagon/somatostatin/pancreatic polypeptide cocktail (GCG/SOM/PP) (green), and HO in human pancreas (B). Scale bars, 50 μm.
Figure 2.
Figure 2.
Treatment of human islets with SB significantly increases β-cell number and function. A–F, Human islets were treated with SB for 24 hours, followed by staining for Ki-67, or TUNEL, or cleaved casp3. The control human islets received DMSO, the solvent for SB (control, CTL). A and B, Percentage of Ki-67+ β-cells by quantification (A) and by representative confocal images (B). C and D, Percentage of TUNEL+ β-cells by quantification (C) and by representative confocal images (D). E and F, Percentage of casp3+ β-cells by quantification (E) and by representative confocal images (F). A positive cell is shown in higher magnification in an inset. G, MTT assay. H, Secreted C-peptide in the culture media. *, P < .05. NS, nonsignificant. n = 5. Scale bars, 10 μm.
Figure 3.
Figure 3.
Molecular mechanisms underlying SB-induced increases in human β-cell proliferation. Representative Western blottings and quantification for phosphorylated TGFbRI (pTGFbRI) and TGFbRI (A), phosphorylated SMAD2 (pSMAD2) and SMAD2 (B), phosphorylated SMAD3 (pSMAD3) and SMAD3 (C), SMAD7 (D), p27 (E), CyclinD1 (F), CyclinD2 (G), CDK4 (H), Cytochrome C (I), Caspase 9 (J), and Beclin-1 (K). GAPDH is a loading control in D–K. SB, SB-treated human islets; CTL, control human islets treated with DMSO. *, P < .05. NS, nonsignificant. n = 5.
Figure 4.
Figure 4.
Pretreatment of human islets with SB improves the outcome of human islet transplantation through augment of β-cell proliferation and function. A, A subtherapeutic number (150) of human islets were pretreated with SB or DMSO as a control for 24 hours. These islets were then transplanted under the kidney capsule of diabetic NOD/SCID mice (10 each) that had received ALX injection 7 days before. Three days after transplantation, 5 mice in each group were killed for analysis of β-cell proliferation in the graft, and the other 5 mice in each group were followed for 28 days and then analyzed for IPGTT and graft insulin. During the process, fasting blood glucose and serum insulin were examined. B, Fasting glucose. C, IPGTT at 28 days after transplantation. D, Serum insulin at day 3 and day 28. E, Graft insulin at day 28 after islet transplantation. F and G, Graft vessel density based on CD31, by quantification (F) and representative images (G). H and I, Percentage of Ki-67+ β-cells in the graft 3 days after transplantation, by quantification (H) and by representative images (I). Yellow arrows pointed to Ki-67+ β-cells. *, P < .05. n = 5. Scale bars, 50 μm. 150 islets, mice transplanted with 150 human islets; 150 SB-islets, mice transplanted with 150 SB-treated human islets.

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