Lung-Derived Microscaffolds Facilitate Diabetes Reversal after Mouse and Human Intraperitoneal Islet Transplantation

Abstract

There is a need to develop three-dimensional structures that mimic the natural islet tissue microenvironment. Endocrine micro-pancreata (EMPs) made up of acellular organ-derived micro-scaffolds seeded with human islets have been shown to express high levels of key beta-cell specific genes and secrete quantities of insulin per cell similar to freshly isolated human islets in a glucose-regulated manner for more than three months in vitro. The aim of this study was to investigate the capacity of EMPs to restore euglycemia in vivo after transplantation of mouse or human islets in chemically diabetic mice. We proposed that the organ-derived EMPs would restore the extracellular components of the islet microenvironment, generating favorable conditions for islet function and survival. EMPs seeded with 500 mouse islets were implanted intraperitoneally into streptozotocin-induced diabetic mice and reverted diabetes in 67% of mice compared to 13% of controls (p = 0.018, n = 9 per group). Histological analysis of the explanted grafts 60 days post-transplantation stained positive for insulin and exhibited increased vascular density in a collagen-rich background. EMPs were also seeded with human islets and transplanted into the peritoneal cavity of immune-deficient diabetic mice at 250 islet equivalents (IEQ), 500 IEQ and 1000 IEQ. Escalating islet dose increased rates of normoglycemia (50% of the 500 IEQ group and 75% of the 1000 IEQ group, n = 3 per group). Human c-peptide levels were detected 90 days post-transplantation in a dose-response relationship. Herein, we report reversal of diabetes in mice by intraperitoneal transplantation of human islet seeded on EMPs with a human islet dose as low as 500 IEQ.

Fig 1. Pilot study of endocrine micro-pancreata (EMPs) implanted subcutaneously.

(A) Dithizone stained EMP showing numerous human islets (red) seeded on the microscaffold. (B) Real time PCR gene expression of insulin and PDX-1 normalized to house-keeping gene TBP. Values are presented as fold expression per cell compared the values obtained from fresh islets (n = 3, single analysis of pooled EMPs removed from three test animals). (C) Average non-fasting blood glucose levels for the three EMP doses implanted subcutaneously. Data are presented as mean ± SEM. This part of the study was completed at the Hebrew University of Jerusalem by or collaborators.

Fig 2. Long-term graft function after mouse islet transplantation.

(A) The proportion of animals that achieved normoglycemia. Normoglycemia was restored in 6 animals from the EMP group compared to 1 from the IP group. This difference was statistically significant (P = 0.0183, Log-rank, Mantel-Cox test). All animals from the kidney capsule group were restored to normoglycemia compared to EMP and IP groups (P< 0.01 & 0.0001 respectively, Log-rank, Mantel-Cox test). Transplanted islets were from five different mouse isolations (n = 30 pancreata per isolation). (B) Average non-fasting blood glucose levels for kidney capsule group (KC500), intraperitoneal free islets group (IP500) and seeded microscaffold group (EMP500). Data are presented as mean ± SEM (one-sided error bars for clarity).

Fig 3. IPGTTs of the transplanted mouse islets six weeks post-transplantation.

Blood glucose measurements after dextrose bolus (A) and AUC analysis (B) did not differ between Naïve (n = 5) and EMP (n = 6) groups (p> 0.05, One way ANOVA with Tukey’s post hoc test). Animals that received free intraperitoneal islets (n = 8) were intolerant to glucose challenge compared to EMP, Naïve and KC (n = 9) groups (*p < 0.05, *** p< 0.001, and ****p< 0.0001 respectively; One way ANOVA with Tukey’s post hoc test). All mice received 3 g/kg 25% dextrose i.p. bolus for this test and blood glucose measurements were taken at t = 0, 15, 30, 60, 90 and 120 min. Data are presented as mean ± SEM.

Fig 4. Histological analysis of explanted islet grafts 60 days post-transplantation.

(A) Mason’s trichrome staining of a cross-section of explanted EMP showing mouse islets of normal structure and size with surrounding background of collagen (blue), smooth muscles, erythrocytes (red) and scaffold-liver interface at (100x). (C) Mason’s trichrome staining of mouse islets seeded on EMP at higher magnification (200x) showing erythrocyte filled blood vessel (arrow) with fluorescent staining of the same sections (B&D) to confirm the presence of insulin (red) and neovascularization (arrows) with positive anti-CD31 staining.

Fig 5. Long-term graft function after human islet transplantation.

(A) The proportion of animals that achieved normoglycemia from the high dose EMP group C was significantly higher (p = 0.0401) compared to the low dose EMP group A. However, this difference was not significant compared to the intermediate dose EMP group B and historical kidney capsule groups (p = 0.671 and 0.889; respectively). (B) Average non-fasting blood glucose levels for all the groups were inversely proportional to transplanted islet dose. (C) Average stimulated human C-peptide levels for Group C was significantly higher than Groups A and B (p<0.001 and p<0.01, respectively; one way ANOVA with Tukey’s multiple comparison test).

Fig 6. Histological analysis of explanted islet grafts 90 days post-transplantation.

(A) Hematoxylin and eosin staining of a cross-section of an explanted EMP graft on the surface of the liver showing multiple islets with an erythrocyte filled blood vessels (arrow). (B) Immunohistochemistry staining of the same section confirming the presence of insulin (red) and glucagon (green).