Targeted mapping and utilization of the perihepatic surface for therapeutic beta cell replacement and retrieval in diabetic non-human primates

Abstract

Introduction: Successful diabetes reversal using pancreatic islet transplantation by various groups illustrates the significant achievements made in cell-based diabetes therapy. While clinically, intraportal islet delivery is almost exclusively used, it is not without obstacles, including instant blood-mediated inflammatory reaction (IBMIR), relative hypoxia, and loss of function over time, therefore hindering long-term success. Here we demonstrate the perihepatic surface of non-human primates (NHPs) as a potential islet delivery site maximizing favorable characteristics, including proximity to a dense vascular network for adequate oxygenation while avoiding IBMIR exposure, maintenance of portal insulin delivery, and relative ease of accessibility through minimally invasive surgery or percutaneous means. In addition, we demonstrate a targeted mapping technique of the perihepatic surface, allowing for the testing of multiple experimental conditions, including a semi-synthetic hydrogel as a possible three-dimensional framework to improve islet viability.

Methods: Perihepatic allo-islet cell transplants were performed in immunosuppressed cynomolgus macaques using a targeted mapping technique to test multiple conditions for biocompatibility. Transplant conditions included islets or carriers (including hydrogel, autologous plasma, and media) alone or in various combinations. Necropsy was performed at day 30, and histopathology was performed to assess biocompatibility, immune response, and islet viability. Subsequently, single-injection perihepatic allo-islet transplant was performed in immunosuppressed diabetic cynomolgus macaques. Metabolic assessments were measured frequently (i.e., blood glucose, insulin, C-peptide) until final graft retrieval for histopathology.

Results: Targeted mapping biocompatibility studies demonstrated mild inflammatory changes with islet-plasma constructs; however, significant inflammatory cell infiltration and fibrosis were seen surrounding sites with the hydrogel carrier affecting islet viability. In diabetic NHPs, perihepatic islet transplant using an autologous plasma carrier demonstrated prolonged function up to 6 months with improvements in blood glucose, exogenous insulin requirements, and HbA1c. Histopathology of these islets was associated with mild peri-islet mononuclear cell infiltration without evidence of rejection.

Discussion: The perihepatic surface serves as a viable site for islet cell transplantation demonstrating sustained islet function through 6 months. The targeted mapping approach allows for the testing of multiple conditions simultaneously to evaluate immune response to biomaterials at this site. Compared to traditional intraportal injection, the perihepatic site is a minimally invasive approach that allows the possibility for graft recovery and avoids IBMIR.

Keywords: beta cell replacement; biomaterials; engraftment; hydrogels; islet transplantation; perihepatic surface; transplantation site; type 1 diabetes mellitus.

Figure 1

Biomaterial targeted mapping evaluation. (A) Phase-contrast image of parallel microchannels in capillary alginate hydrogel used in targeted mapping. (B and C) Biocompatibility and general safety of approach was assessed in non-diabetic NHPs. Weight trend with overall mean and standard deviation is presented.

Figure 2

Overview of PH surface islet transplantation and targeted mapping technique. (A) Representative schematic of targeted mapping technique using the left lateral liver lobe. Numbers correspond to different map site constructs spatially distributed by grid. (B) Islet-carrier constructs for each map site for each recipient. Islet dose presented in islet equivalents per kilogram. CAG, capillary alignate hydrogel; Plasma, autologous plasma (C) Left. Liver at necropsy showing islet grafts on PH surface. Grafts are circled in black. Right. H&E staining of representative islet-capillary alginate hydrogel map site, black arrows point to areas of aggregated hydrogel. (D) Study design overview for NHP recipients including immunosuppression protocol and time of graft retrieval. (E) Reverse Kaplan–Meier estimate of time to islet engraftment in NHPs calculated from the date of transplantation to the date of engraftment as measured by C-peptide ≥0.5 ng/ml. (F) Daily measures of preprandial (solid line) and postprandial glucose (dashed line) in mg/dl and exogenous insulin requirements (gray) in U/kg with inset showing human C-peptide (ng/ml) measured randomly (green), under fasting (blue), or stimulated (yellow) conditions by days post-transplant in recipient 16JP3 with a dose of 6,400 IE/kg, (G) in recipient 16JP11 with a dose of 6,687 IE/kg, and (H) in recipient 16JP14 with a dose of 14,788 IE/kg, low purity. (I) Insulin immunohistochemistry staining for islets in recipients 16JP3, (J) 16JP11, and (K) 16JP14.

Figure 3

Representative histology of graft site in a non-diabetic, targeted mapping NHP recipient. Sections taken from the site injected with islet-capillary alginate hydrogel construct at 4× (top) and 10× (bottom) magnification with various stains including (A) insulin staining, (B) CD31 IHC staining for endothelial lined blood vessels, (C) Iba-1 IHC staining for macrophages, with black arrows pointing to the areas of background staining by capillary alginate hydrogel, (D) CD3 IHC staining for T-cells, (E) CD20 IHC, and (F) CD79a staining for B-cells. Scale bar: 500 µm at 4× magnification; 100 µm at 10× magnification.

Figure 4

Metabolic effects of PH surface islet transplant in diabetic, immunosuppressed NHP recipients. (A) HbA1c % measured at transplant and up until graft retrieval for 16JP3, (B) 16JP11, and (C) 16JP14. (D) IVGTT measurements taken after STZ (blue), 28 (orange), 78 (black), and 126 (green) days after transplant for 16JP3, (E) after STZ (blue), 28 (orange), 77 (black), and 125 (green) days after transplant for 16JP11, (F) after STZ (blue) and 43 (orange) days after transplant for 16JP14. KG, glucose disappearance rate (%/min). Daily weights for (G) 16JP3, (H) 16JP11, and (I) 16JP14. Mean weight and standard deviation presented within the graph.

Figure 5

Representative histology of graft site in a diabetic NHP recipient. Sections taken from the site injected with islet-autologous plasma at 10× magnification with various stains including (A) H&E, (B) Iba-1 IHC staining for macrophages, (C) CD3 IHC staining for T-cells, (D) CD20 IHC staining for B-cells, (E) CD31 IHC staining for endothelial lined blood vessels, and (F) β3 tubulin IHC staining for neurons. The dashed line highlights the cluster of islets. Scale bar 100 µm.