Table 1.
Growth factor concentrations in Matrigel and growth factor-reduced Matrigel.
[24]
Fig 1.
Condition of intraportally and intramuscularly transplanted islets at POD3.
Substantial inflammatory infiltrates adjacent to intramuscularly transplanted islets with hemorrhage around islets were observed. Transplanted islets were observed as clustered aggregates with necrotic and apoptotic islet cells at the center of clusters. Necrotic and apoptotic cells were rarely observed following intrahepatic islet transplantation. Scale bar, 100 μm.
Table 2.
Proportion of mice achieving normoglycemia following islet transplantation and MD50 values for each transplant site.
Fig 2.
Islet residual rate, viability, and stimulation index following culture with or without Matrigel.
Islets were cultured for 96 h with or without Matrigel before the assessment of residual rate, viability, and the ratio of insulin released in response to stimulation by high and low concentration glucose solutions (SI). (A) The residual rate of islets in the GFR Matrigel or Matrigel groups was significantly higher than that in the islets-only group. (B) Islet cell viability in GFR Matrigel or Matrigel groups was significantly higher than that in the islets-only group. Right figures; green: viable cells, red: dead cells. Scale bar, 100 μm. (C) The SI was significantly higher in the GFR Matrigel and Matrigel groups compared with the islets-only group. *P < 0.05. GFR, growth factor reduced; SI, stimulation index.
Fig 3.
Intracellular signaling activity in islets cultured in Matrigel.
FAK, Erk, and Akt protein levels in islets were evaluated by Western immunoblotting to assess the effect of Matrigel on cellular growth and cytoprotection. Cell lysates obtained from the islets-only, Matrigel, and GFR Matrigel groups were subjected to SDS-PAGE and Western immunoblotting using the indicated antibodies. Glyceraldehyde, phosphate dehydrogenase was used as the loading control. Phosphorylated FAK, Erk, and Akt were strongly expressed in the Matrigel and GFR Matrigel groups compared with the islets-only group. Erk, extracellular signal-regulated kinase; FAK, focal adhesion kinase; GFR, growth factor-reduced; SDS, sodium dodecyl sulfate.
Fig 4.
Changes in the blood glucose concentrations, serum insulin level, and IPGTT following transplantation.
Transplantation outcomes were evaluated by the measurement of BG and serum insulin levels and GTTs. (A) BG levels were significantly improved in islets embedded in ECM (both Matrigel and GFR Matrigel groups) compared with the islets-only group. BG levels were significantly lower in the Matrigel group compared with the GFR Matrigel group at POD 28. (B) Comparison of changes in blood glucose concentrations between intramuscular transplantation in the Matrigel group and the intraportal transplantation group. No significant differences were observed in changes in blood glucose concentration between intramuscular transplantation in the Matrigel group and the intrahepatic transplantation group. (C) Serum insulin levels were significantly increased in the GFR Matrigel and Matrigel groups compared with the islets-only group, and levels in the Matrigel were significantly higher than those in the GFR Matrigel group at POD 28. (D) IPGTT AUC values in both the GFR Matrigel and Matrigel groups were significantly lower than those in the islets-only group. AUC, area under the curve; BG, blood glucose; ECM, extracellular matrix; GFR, growth factor reduced; GTT, glucose tolerance test; IPGTT, intraperitoneal glucose tolerance test; POD, postoperative day.
Fig 5.
Intramuscular transplanted islets at POD 3.
A. A large number of inflammatory infiltrates (yellow circle) were observed surrounding islets with a number of islets found to have underwent necrosis in the islets-only group. B. Islets in the GFR Matrigel and Matrigel groups were separated from surrounding inflammatory cells by extracellular matrix. Few necrotic islets were observed in the Matrigel groups. Upper panel, hematoxylin and eosin. Lower panel, insulin immunohistochemistry. Scale bar, 100 μm. POD, postoperative day.
Fig 6.
TUNEL and Ki67 immunohistochemistry of intramuscularly transplanted islets at POD 3.
(A) The proportion of TUNEL-positive cells (arrows) per islet was significantly lower in the GFR Matrigel and Matrigel groups compared with the islets-only group. The proportion of TUNEL-positive apoptotic cells was 7.50 ± 1.79 in the islets-only group, 1.37 ± 0.65 in the GFR Matrigel group, and 1.01 ± 0.41 in the Matrigel group (P = 0.0014 for islets-only vs. GFR Matrigel; P = 0.0024 for islets-only vs. Matrigel). (B) The proportion of Ki67-positive cells (arrows) per islet was significantly higher in the GFR Matrigel and Matrigel groups than that in the islets-only group. The proportion of Ki67-positive cells per islet was 1.38 ± 0.43 in the islets-only group, 3.80 ± 0.55 in the GFR Matrigel group, and 5.58 ± 1.02 in the Matrigel group (P = 0.001 for islets-only vs. GFR Matrigel; P = 0.00025 for islets-only vs. Matrigel). Scale bar, 100 μm GFR, growth factor reduced; TUNEL, TdT-mediated deoxyuridine triphosphate-biotin nick end labeling.
Fig 7.
CD31 staining of intramuscularly transplanted islets at POD 14.
(A) Assessment of neovessels directly contiguous with islets. The number of CD31-positive microvessels (arrows) adjacent to islets was significantly higher in the Matrigel group than in the islets-only and GFR Matrigel groups. The number of microvessels adjacent to islets was 4.68 ± 0.47 in the islets-only group, 5.75 ± 0.75 in the Matrigel group, and 8.61 ± 0.75 in the GFR Matrigel group (P = 0.0001 for islets-only vs. Matrigel; P = 0.012 for GFR Matrigel vs. Matrigel). (B) Assessment of islet revascularization. No significant difference in the number of microvessels was observed among the three groups. Scale bar, 100 μm GFR, growth factor reduced; POD, postoperative day.